CN103561730A

CN103561730A - Cis 3,4-dihydroxy-2-(3-methylbutanoyl)-5-(-3-methylbutyl)-4-(4-methylpentanoyl)cyclopent-2-en-1-one derivatives, substantially enantiomerically pure compositions and methods

Info

Publication number: CN103561730A
Application number: CN201180063785.1A
Authority: CN
Inventors: 布莱恩·卡罗尔; 加里·达兰德; 阿奴拉·德赛; 维拉·康达; ***·J·达尔伯格; 简·厄班
Original assignee: KinDex Therapeutics LLC
Current assignee: KinDex Therapeutics LLC
Priority date: 2010-10-30
Filing date: 2011-10-28
Publication date: 2014-02-05
Also published as: ES2829388T3; CN110215442A; JP2013542225A; EP2632448B1; US8410178B2; AU2011320140B2; JP6259287B2; US20130018105A1; US20120108671A1; CA2853974A1; KR20140027910A; AU2011320140C1; WO2012058649A1; CN110200955A; EP2632448A1; AU2011320140A1; US8410179B2; JP2017101019A; US20130217781A1; EP2632448A4

Abstract

The present application provides cis 3,4-dihydroxy-2-(3-methylbutanoyl)-5-(3- methylbutyl)-4-(4-methylpentanoyl)cyclopent-2-en-1-one derivatives and substantially enantiomerically pure compositions thereof. These derivatives include (+)-(4S,5R)- 3,4- dihydroxy-2-(3-methylbutanoyl)-5-(3-methylbutyl)-4-(4-methylpentanoyl)cyclopent-2-en-1-one, (-)-(4R,5S)-3,4-dihydroxy-2-(3-methylbutanoyl)-5-(3-methylbutyl)-4-(4- methylpentanoyl)cyclopent-2-en-1-one, and salts and crystals thereof. The application further provides methods of using the disclosed compounds and compositions to activate PPARgamma, activate GPR120, inhibit inflammation, and treat conditions responsive to PPARgamma modulation, conditions responsive to GPR120 modulation, and metabolic disturbances such as diabetes.

Description

Cis 3,4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone derivatives, compositions and the method for enantiomer-pure substantially

related application

The application requires the right of the U.S. Provisional Application 61/448,150 of U.S. Provisional Application submission March 1 in 61/408,572,2011 and the U.S. Provisional Application 61/508,434 of submission on July 15th, 2011 of submission on October 30th, 2010.The disclosure of these provisional application comprises that by quoting accompanying drawing full text adds herein.

Background technology

The abundantest member in alpha acid family is (-)-.alpha.-bitter acid.Reported the absolute configuration (De Keukeleire1970) of (-)-.alpha.-bitter acid before, and definite (the De Keukeleire1971) of the absolute configuration of the cis being obtained by (-)-.alpha.-bitter acid and trans iso-alpha acid.

To the reduzate of so-called ' the iso-alpha acid of reduction '-obtained by cis and trans iso-alpha acid-further sort out.This additional position and saturation that depends on saturated bond of sorting out.The classes of compounds obtaining by only C6 carbonyl reduction being become to hydroxyl is collectively referred to as the iso-alpha acid of rho (RIAAs).The iso-alpha acid of tetrahydrochysene (THIAA) class totally refers to that only two prenyls are those saturated compounds.Similarly, six hydrogen-iso-alpha acid (HIAA) totally refers to the reductive derivative of iso-alpha acid, and it is included in the saturated of the hydroxyl of C6 and two isoprene base section.Because various short-chain fatty acid participate in the biosynthesis pathway of corresponding phloroglucinol (phlorglucinol), except the existence of cis and trans diastereomer, in every class of this three apoplexy due to endogenous wind, also have various congeners (Wang2008).Phloroglucinol class is the conventional precursor as the alpha acid of iso-alpha acid precursor, this iso-alpha acid then be the precursor of the iso-acid of α of reduction.

Recently the iso-alpha acid that has shown reduction has beneficial effect in various external and murine models in the treatment of obesity, dyslipidemia and inflammation.The Hun He Wu – of the congener of the iso-alpha acid of rho and stereoisomer is called RIAA – and presents in the anti-inflammatory activity by the ripe 3T3-L1 adipose cell of tumor necrosis factor α (TNF-α)-stimulation and the lipid in differentiation 3T3-L1 adipose cell and form activity (Babish2010).

The Hun He Wu – of the congener of the iso-alpha acid of tetrahydrochysene and stereoisomer is called THIAA – and suppresses in vitro spleen tyrosine kinase (Syk), bruton's tyrosine kinase (Btk), phosphatidyl-inositol 3-kinase (PI3-kinases) and the activity of GSK3 β (GSK3 β) and the phosphorylation of beta-catenin.In addition, THIAA suppresses osteoclast and generates, the prostaglandin E that TRAP is active and repressed IL-1 β – activates that lowers and lower to the conversion of osteoclast as RAW264.7 cell ₂, and RASF matrix metalloproteinase 3, IL-6, IL-8 and MCP 1 indicated.In addition,, in suffering from the mice of collagen-induced arthritis (CIA),, this equal mixture of congener and stereoisomer (that is THIAA) reduces significantly arthritis index and reduces bone, joint and cartilage degradation.Blood serum IL-6 concentration when processing with THIAA in these mices is also with dosage-dependent mode suppressed (Konda2010).

Consistent with these results of study, also reported that RIAA and THIAA are suppressed at respectively the prostaglandin E in the RAW264.7 macrophage that is subject to lipopolysaccharide-stimulation ₂(PGE ₂) produce and suppress derivable COX-2 (COX-2) protein expression.In addition, each mixture, that is, RIAA and THIAA, reported respectively in dose dependent mode and lowered NF-κ B core transposition and abundance (Tripp2009).

The heterogeneity of RIAA and THIAA hinders various congeners and stereoisomer-be present in RIAA or THIAA mixture-with respect to their understanding and the understanding individual and even relation between biological activity relatively.In addition, be present in synergistic probability between the multiple compounds in RIAA and THIAA and can largely, if not fully, explain the viewed biological activity of these mixture.

In order to understand and understand various congeners and stereoisomer with respect to the relation between their individual and even relative biological activity, it is essential that the compound form with pure and mild enantiomer-pure substantially obtains the compound of being paid close attention to, and measure their individual biological activity with the form of pure and mild enantiomer-pure substantially.This necessity must be that the biological activity of any heterogeneous material (for example, THIAA, RIAA and HHIAA) of the mixture from comprising different molecular depends on the conclusion that percentage ratio composition, spatial chemistry, structure and other character of the different molecular that forms this heterogeneity material draw.For those reasons, exist preparation and purification for the needs of the iso-alpha acid derivant of the reduction of the enantiomer-pure substantially of pharmaceutical compositions and treatment.

Except this needs, also need to produce the compound of pure and mild enantiomer-pure substantially to be suitable for the form of the conventional various steps that face in drug development.Being prepared with of the pure crystal form of potential drug candidate is beneficial to drug development.The chemistry that advantage is drug candidate and the improved sign of physical property.For crystal form, conventionally there is the pharmacokinetics more favourable than amorphous forms, and they are easier to processing conventionally.Improved storage stability is conventionally another advantage relevant to crystal form.The intrinsic physical property of crystal form of potential drug candidate is the key factor when selecting specific active constituents of medicine.Example is that potential medicine is formulated in applicable compositions for the production of, storage and consumption.Particularly, before grinding and afterwards, the flowable of crystalline solid affects the mode of drug candidate of how processing in processing and medicament production process greatly.At the granule of polished solid form, do not allow in runny situation, pharmaceutical preparation researcher will try hard to developing drugs preparation to make up this difficulty.This generally includes for example use of silica sol, Talcum and starch or alkali calcium phosphate of fluidizer.The another kind of solid state properties of potential medical compounds is its dissolution velocity in moisture fluid.The physical property relevant to specific crystalline polymorph is because the spatial orientation of each and every one molecule of the structure cell of this crystalline polymorph of composition and unique conformation inherently.Specific crystalline polymorph has the thermal property that is generally different from amorphous forms or another polytypic uniqueness.

Polytypic thermal property utilizes for example capillary melting point, thermogravimetry (TGA) and differential scanning calorimetry (DSC) to measure.The result of these measurements is used for distinguishing and identifying the existence of polymorph form and distinguish them each other.Specific polymorph form generally has by including but not limited to the detectable different crystallographic properties of various technology and the spectral quality of X-ray powder diffraction (XRPD), single-crystal x-radiocrystallgraphy and infrared spectrometry.

Summary of the invention

In this article; cis 3 is provided in some embodiments, and 4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone (" KDT500 ") derivant and the compositions of enantiomer-pure and the pharmaceutical compositions that comprises these derivants substantially.

In some embodiments; the KDT500 derivant providing is herein selected from (+)-(4S with structure shown in formula I; 5R)-3; 4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone (" (+)-KDT500 "), has (-)-(4R of structure shown in formula II; 5S)-3; 4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone (" (-)-KDT500 "), with and salt and crystallization.

formula I

formula II

In some embodiments, the KDT500 derivant providing is herein the salt of (+)-KDT500 or (-)-KDT500.In some embodiments, these derivants can be inorganic salt or the organic salt of (+)-KDT500 or (-)-KDT500, include but not limited to potassium salt, aluminum salt, calcium salt, mantoquita, guanidinesalt, iron salt, lithium salts, magnesium salt, sodium salt, zinc salt, cinchonidine salt, cinchonine salt and diethanolamine salt.In some in these embodiments, described derivant can be the potassium salt (" (+)-KDT501 ") of (+)-KDT500 with structure shown in formula III.

formula III

In some embodiments, the KDT500 derivant providing is herein the crystallization of (+)-KDT500, (-)-KDT500 or its salt.In some of these embodiments, described derivant is the crystallization of (+)-KDT501, and in some of these embodiments, described crystallization has monoclinic system space group P21212, and unit cell dimension is

α=90 °,

β=90 °, and γ=90 °.In some embodiments, described crystallization has one or more in the feature shown in table 2-4.

The compositions of the enantiomer-pure substantially of KDT500 derivant provided herein is provided herein in some embodiments.In some embodiments, these substantially the compositions of enantiomer-pure comprise (+)-KDT500, (-)-KDT500, or its salt or crystallization.

Substantially the pharmaceutical compositions of enantiomer-pure is provided herein in some embodiments, and a kind of KDT500 derivant and one or more pharmaceutically acceptable carriers that provide are herein provided for it.In some embodiments, these substantially the pharmaceutical compositions of enantiomer-pure comprise (+)-KDT500, (-)-KDT500, or its salt or crystallization.

Provide in some embodiments herein by KDT500 derivant one or more or its compositions providing herein to come in vitro or the interior method that causes lipid formation (lipogenesis), initiation lipogenesis (adipogenesis), activates PPAR γ or activation GPR120 of body.In some embodiments, described compositions is the pharmaceutical compositions of enantiomer-pure substantially, and it comprises KDT500 derivant and one or more pharmaceutically acceptable carriers.In some embodiments, described method is used for treating having in this object needing and forms, reduces the active relevant disease of lipogenesis or reduction PPAR γ or GPR120 to minimizing lipid.

A kind of method is provided herein in some embodiments, the method is treated disease that to PPAR γ modulation respond, treatment disease that to GPR120 modulation respond, treatment dysbolismus, inflammation-inhibiting in this object of needing by one or more the KDT500 derivants that provide to object drug treatment effective dose or its compositions herein, or the treatment patient's condition relevant to inflammation.In some in these embodiments, described derivant is the pharmaceutical compositions administration of the enantiomer-pure substantially by providing herein.In some embodiments, the described disease that modulation responds to PPAR γ is type ii diabetes, obesity, hyperinsulinemia, metabolism syndrome, non-alcoholic fatty liver disease, nonalcoholic steatohepatitis, autoimmune disorder or proliferative disorders.In some embodiments, described dysbolismus is diabetes.In some embodiments, the administration of compound provided herein or compositions causes glucose and/or lipid level (comprising triglyceride level) to decline.

accompanying drawing summary

Fig. 1: the stereochemical structure of (+)-KDT501.From structure cell, omit potassium ion and hydrone.

Fig. 2: the ORTEP figure of (+)-KDT501 crystallization to its asymmetric cell.

Fig. 3: the XRPD diffraction pattern of (+)-KDT501.

Fig. 4: the TG/DTA thermogram of (+)-KDT501.

Fig. 5: the impact that (+)-KDT501 forms lipid in 3T3-L1 adipose cell.Rosiglitazone is as positive control.

Fig. 6: the impact of (+)-KDT501 on the MMP-9 expression of TNF-α in THP-1 cell-(A) and LPS-(B) mediation.Data representation is from the meansigma methods ± SD of 4 tests.

Fig. 7: the impact of (+)-KDT501 on the IL-1 β expression of TNF-α in THP-1 cell-(A) and LPS-(B) mediation.Data representation is from the meansigma methods ± SD of 4 tests.

Fig. 8: the impact of (+)-KDT501 on the MCP-1 expression of TNF-α in THP-1 cell-(A) and LPS-(B) mediation.Data representation is from the meansigma methods ± SD of 4 tests.

Fig. 9: the impact of (+)-KDT501 on the RANTES expression of TNF-α in THP-1 cell-(A) and LPS-(B) mediation.Data representation is from the meansigma methods ± SD of 4 tests.

Figure 10: the impact of (+)-KDT501 on the MIP-1 alpha expression level of TNF-α in THP-1 cell-(A) and LPS-(B) mediation.Data representative is from the meansigma methods ± SD of 4 tests.

Figure 11: the PGE of (+)-KDT501 to IL-1 β-mediation in RASFs ₂the impact of level.

Figure 12: the impact of (+)-KDT501 on the MMP13 level of IL-1 β-mediation in RASFs.

Figure 13: the impact of (+)-KDT501 on PPAR gamma activity.Rosiglitazone and telmisartan are as positive control.

Figure 14: the impact of (+)-KDT501 on PPAR alpha active.GW590735 is used as negative control as positive control and rosiglitazone.

Figure 15: the impact of (+)-KDT501 on PPAR δ activity.GW0742 is used as negative control as positive control and rosiglitazone.

Figure 16: (+)-KDT501 and the impact of (-)-KDT501 on PPAR gamma activity.Rosiglitazone is as positive control.

Figure 17: the impact of (+)-KDT501 on GPR120 activity.Data represent the mean+/-standard deviation of replication.DHA, EPA and α-LA are with comparing.

Figure 18: (+)-KDT501 and (-)-KDT501 impact on DAPK1 activity in acellular test.

The impact of Figure 19: KDT501 on the blood sugar level in ZDF rat.

The impact of Figure 20: KDT501 on blood triglyceride level in ZDF rat.

The impact of Figure 21: KDT501 on blood sugar level in DIO mice.

The impact of Figure 22: KDT501 on blood insulin levels in DIO mice.

The impact of Figure 23: KDT501 on HbA1C level in DIO mice.

The impact of Figure 24: KDT501 on fat mass in DIO mice.

The specific embodiment

The following description of the present invention is only intended to illustrate several embodiment of the present invention.Therefore the concrete change of, discussing should not be interpreted as limitation of the scope of the invention.To those skilled in the art, obviously easily see without departing from the present invention and can carry out various equivalences, variation and modification, and the equivalent like this embodiment of understanding should be included in herein.

As disclosed herein, cis 3,4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone (" KDT500 "), and derivant is synthesized, Purification and Characterization.Therefore, provide in some embodiments KDT500 derivant and salt thereof and crystallization herein, comprise the crystallization of described salt.Compositions is also provided herein, and it comprises these derivants and salt and crystallization, comprises the compositions of enantiomer-pure substantially.

Term " salt " can refer to the acceptable salt of any pharmacy while using in this article, for example comprise that inorganic base salts is as potassium salt, aluminum salt, calcium salt, mantoquita, guanidinesalt, iron salt, lithium salts, magnesium salt, sodium salt and zinc salt, and organic alkali salt is as cinchonidine salt, cinchonine salt and diethanolamine salt.For example, in Berge J Pharm Sci66:1 (1977), can find other examples of the acceptable salt of pharmacy and prepared product as described in the present invention.

In some embodiments, the KDT500 derivant providing herein has the structure shown in formula I or formula II:

formula I

formula II

The compound of formula I and formula II represents respectively (+)-(4S; 5R)-3; 4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone (being called " (+)-KDT500 " herein) and (-)-(4R; 5S)-3,4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone (being called " (-)-KDT500 " herein).Therefore, some preferred embodiment in, the KDT500 derivant providing is herein (+)-KDT500 or (-)-KDT500.

In some embodiments, the KDT500 derivant providing is herein the salt of KDT500.The salt of KDT500 includes but not limited to potassium salt, magnesium salt, calcium salt, zinc salt, iron salt, sodium salt, mantoquita, guanidinesalt, cinchonidine salt and the cinchonine salt of KDT500.For example; the salt of KDT500 can be ((+)-(4S; 5R)-3; 4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles the potassium salt (being called " (+)-KDT501 " herein) of penta-2-alkene-1-ketone, has the structure shown in formula III:

Formula III

In other embodiments, the KDT500 derivant providing is herein crystallization or its salt of KDT500, comprises for example KDT501.In some of these embodiments, the crystallization of provide (+)-KDT501, it has monoclinic system space group P21212, and unit cell dimension is

(α=90 °),

(β=90 °) and (γ=90 °).This crystal form of (+)-KDT501 is called " crystallization (+)-KDT501 " herein.

In some embodiments, crystallization (+)-KDT501 has bond distance and the angle shown in the three-dimensional atomic coordinates shown in table 2, table 3, and/or the anisotropy displacement parameter shown in table 4, wherein this anisotropy shift factor index adopts this form :-2 π ²[h ²a* ²u ¹¹+ ...+2h k a*b*U ¹²].

Compositions is provided herein in some embodiments, and one or more KDT500 derivants providing are herein provided for it.In some of these embodiments, described compositions is enantiomer-pure substantially.Term " enantiomer-pure substantially " refers to a kind of compositions while using in this article, in said composition 90% or more specific compound be the first enantiomeric form, and 10% or still less be the second enantiomeric form.For example, in (+)-KDT500 compositions of enantiomer-pure substantially, in said composition 90% or more described KDT500 be (+)-KDT500 and 10% or still less be (-)-KDT500.In some embodiments, " first enantiomeric form " of compound comprises salt and the crystallization of this enantiomeric form.For example, in (+)-KDT500 compositions of enantiomer-pure substantially, in said composition 90% or more described KDT500 be (+)-KDT500 form or its salt or crystallization, and 10% or still less be (-)-KDT500 form or its salt or crystallization.In some embodiments, the compositions of enantiomer-pure can comprise 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95% or 99.99% or the first enantiomeric form of more compound substantially.

The compositions of (+)-KDT500 is provided in some embodiments.In some of these embodiments, described compositions is enantiomer-pure substantially.In some of these embodiments, in said composition, (+) of certain percentage-KDT500 is the salt of (+)-KDT500 or the form of crystallization.In some of these embodiments, all (+)-KDT500 in said composition are salt or crystal form.Therefore, the compositions of the enantiomer-pure substantially of (+)-KDT500 salt or crystallization is provided herein.In other embodiments, in said composition, there is no (+)-KDT500 is salt or crystal form.

The compositions of (+)-KDT500 is provided in some embodiments.In some of these embodiments, described compositions is enantiomer-pure substantially.In some of these embodiments, in said composition, (-) of certain percentage-KDT500 is the salt of (-)-KDT500 or the form of crystallization.In some of these embodiments, in said composition, all (-)-KDT500 are salt or crystal form.Therefore, the compositions of the enantiomer-pure substantially of (-)-KDT500 salt or crystallization is provided herein.In other embodiments, there is no (-)-KDT500 be salt or crystal form to said composition.

Pharmaceutical compositions is provided herein in some embodiments, and one or more and one or more pharmaceutically acceptable carriers in KDT500 derivant providing are herein provided for it.In some embodiments, described pharmaceutical compositions is enantiomer-pure substantially.In some of these embodiments, the pharmaceutical compositions of described enantiomer-pure substantially comprises (+)-KDT500, (-)-KDT500 or its salt or crystallization." pharmaceutically acceptable carrier " refers to while using in this article and participates in relevant compound to carry or be transported to from a tissue, organ or the part of health the acceptable material of pharmacy, compositions or the carrier of another tissue, organ or the part of health.Such carrier can comprise, for example, and liquid or solid filler, diluent, excipient, solvent, encapsulating material, stabilizing agent, or their certain combination.Each component of described carrier must be " pharmacy is acceptable ", because it must with other component compatibility of described compositions, and must be suitable for contacting the part of any tissue, organ or the health that may touch, meaning is that it must not have toxicity, stimulation, atopic reaction, immunogenicity or excessively exceed the risk that it treats any other complication of benefit.

Example for the pharmaceutically acceptable carrier of the compositions that provides herein includes but not limited to: (1) saccharide, for example lactose, glucose, sucrose or mannitol; (2) starch based, for example corn starch and potato starch; (3) cellulose and derivant thereof, for example sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) Fructus Hordei Germinatus; (6) gelatin; (7) Talcum; (8) excipient, for example cupu oil and suppository wax; (9) oils, for example Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, safflower oil, Oleum sesami, olive oil, Semen Maydis oil and soybean oil; (10) glycols, for example propylene glycol; (11) polyhydric alcohol, for example glycerol, Sorbitol, mannitol and Polyethylene Glycol; (12) esters, for example ethyl oleate and ethyl laurate; (13) disintegrating agent, for example agar or calcium carbonate; (14) buffer agent, for example magnesium hydroxide and aluminium hydroxide; (15) alginic acid; (16) apirogen water; (17) isotonic saline solution; (18) Ringer's mixture; (19) alcohol, for example ethanol and propanol; (20) phosphate buffered solution; (21) paraffin; (22) lubricant, for example Talcum, calcium stearate, magnesium stearate, solid polyethylene glycol or sodium lauryl sulfate salt; (23) coloring agent; (24) for example silica sol, Talcum and starch or tricalcium phosphate of fluidizer; (24) other are for the compatible material of the non-toxicity of pharmaceutical compositions acetone for example.In one embodiment, at this pharmaceutically acceptable carrier used, be moisture carrier, for example, buffer saline etc.In other embodiments, described pharmaceutically acceptable carrier is polar solvent, for example acetone and alcohol.

The pharmaceutical compositions providing at this also can comprise the acceptable required complementary material of physiological status that approaches of one or more pharmacy.For example, compositions can comprise one or more pH adjusting agents, buffer agent or toxicity regulator, comprises such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate etc.

The pharmaceutical compositions providing herein can be formulated into applicable dosage form, comprise, for example capsule, cachet, pill, tablet, containing agent, (use has the base material of seasoning, usually sucrose and arabic gum or Tragacanth), powder, granule, the solution in moisture or water-free liquid or suspensoid, oil-in-water or Water-In-Oil liquid emulsion, elixir or syrup, or lozenge (is used inertia base material, for example gelatin and glycerol, or sucrose and arabic gum) and/or collutory etc., every kind of KDT500 derivant that comprises scheduled volume is as active component.In some embodiments, described compositions can be formulated into time controlled released delivery vector, for example Gyrocap." time controlled released carrier " refers to any delivery vector of instant-free active component when a period of time rather than administration while using in this article.In other embodiments, described compositions can be formulated into rapid release delivery vector.

Tablet can, by compacting or molding, optionally be prepared together with one or more auxiliary elements.The tablet of compacting (for example can be used binding agent, gelatin or hydroxypropyl cellulose), lubricant, inert diluent, antiseptic, disintegrating agent (for example, Explotab or crosslinked sodium carboxymethyl cellulose), surfactant or dispersant be prepared.The KDT500 derivant that molding tablet can be pulverized by molding in applicable machine or with inert liquid diluent further the mixture of moistening enantiomer-pure be substantially prepared.Tablet, and other solid dosage formss, for example dragee, capsule, pill and granule, optionally by indentation, or with for example known other coatings preparations in enteric coating and field of pharmaceutical preparations of coating or shell.They also can be formulated to provide slow release or the controlled release of KDT500 derivant, wherein use, and for example, the hydroxypropyl emthylcellulose of different proportion is to provide the release profiles of expectation, other polymeric matrixs, liposome and/or microsphere.They can be for example, by antibacterial-hold back filter to filter, or sterilizes by added immediately the antibacterial of the aseptic solid composite form of the medium that dissolves in sterilized water or some other sterile injectable before using.These compositionss also can be optionally to comprising delustering agent (pacifying agent), and can have them and only or preferably in a gastrointestinal part, optionally in the mode postponing, discharge the compositions of KDT500 derivant.The example of spendable embedding composition (embedding composition) comprises polymer material and wax.Described KDT500 derivant can also be the form of micro encapsulation, if suitably, have one or more above-mentioned excipient.

In compositions provided herein, the concentration of KDT500 derivant can change.Can, according to the needs of concrete selected mode of administration and biosystem, on the basis of fluid volume, viscosity, body weight etc., select concentration.In some embodiments, in compositions provided herein, the concentration of KDT500 derivant can be approximately 0.0001% to 100%, approximately 0.001% to approximately 50%, approximately 0.01% to approximately 30%, approximately 0.1% to approximately 20%, or approximately 1% to about 10%wt/vol.

The method of analysis, synthetic, purification and/or crystallization KDT500 derivant provided herein is also provided herein, and the method for the KDT500 of the enantiomer-pure substantially compositions that provides herein of analysis, crystallization, purification and/or crystallization.

In some embodiments, the synthetic method providing herein produces single enantiomer of KDT500 derivant.For example, described synthetic method can produce only (+)-KDT500 or only (-)-KDT500.In other embodiments, described synthetic method produces the mixture of the enantiomeric form of KDT500 derivant.In these embodiments, can carry out a step or multistep later separation and/or purification step with separation list enantiomeric form or the compositions of the enantiomer-pure substantially provide is herein provided.

In some of the synthetic and purification process providing in this article, (+)-KDT500 and/or (-)-KDT500 can synthesize (for example, referring to,, following examples 11) as raw material with .beta.-bitter acid.In one of these synthetic/purification process, first .beta.-bitter acid is converted to tetrahydrochysene-deoxidation .alpha.-bitter acid by reduction-dealkylation, and tetrahydrochysene-deoxidation .alpha.-bitter acid is converted to the tetrahydrochysene-.alpha.-bitter acid ((+) and/or (-)) of enantiomer-pure by asymmetric oxidation.The enantiomer of gained provides enantioselectivity and cis-selectivity isomerization to be purified and to change into the iso-.alpha.-bitter acid of corresponding cis.

In another embodiment, (+)-KDT500 and/or (-)-KDT500 can synthesize (for example, referring to,, following examples 12) as raw material with deoxidation .alpha.-bitter acid.In one embodiment, first deoxidation .alpha.-bitter acid is changed into respectively (+)-.alpha.-bitter acid and/or (-)-.alpha.-bitter acid by asymmetric oxidation.(+)-.alpha.-bitter acid is converted to (-)-cis IsoHopCO2N. of corresponding enantiomer-pure by enantioselectivity and cis-selectivity isomerization, then by hydrogenation, be further converted to (-)-KDT500.(-)-.alpha.-bitter acid is converted to (+)-cis IsoHopCO2N. of corresponding enantiomer-pure by enantioselectivity and cis-selectivity isomerization, then by hydrogenation, be further converted to (+)-KDT500.

In another embodiment, (+) of enantiomer-pure-.alpha.-bitter acid and (-)-.alpha.-bitter acid can be from raceme (±)-.alpha.-bitter acid purification (for example, referring to,, following examples 13).In another embodiment, (+) of enantiomer-pure-tetrahydrochysene .alpha.-bitter acid and (-)-tetrahydrochysene .alpha.-bitter acid can be from raceme (±)-tetrahydrochysene .alpha.-bitter acid purification (for example, referring to,, following examples 14).

As disclosed in this article, (+)-KDT501 is evaluated lipid formation and adipogenic impact in 3T3-L1 mouse fibroblast cell model.Observe (+)-KDT501 all causes lipid in dosage-dependent mode and forms and lipogenesis.3T3-L1 mouse fibroblast cell can be studied front adipose cell breeding, Adipocyte Differentiation and insulin sensitizing agent effect (Fasshauer2002; Li2002; Raz2005).The reagent of having reported the lipid picked-up promoting in adipose cell improves insulin sensitivity.To be fatty acid be included into from blood plasma the improvement (Martin1998) that causes the relative minimizing of fatty acid in muscle to have the glucose uptake of following adipose cell to a kind of hypothesis.Insulin desensitization effect at muscle and liver free fatty acid can lower because of thiazolidinedione treatment.These in vitro results are by clinical confirmation (Boden1997; Stumvoll2002).

Evaluate (+)-KDT501 and (-)-KDT501 under the agonist ligand of PPAR γ, SCN2A and AGTR2 exists with the ability of these target competitive bindings.Two kinds of molecules all show under the agonist ligand of target exists in conjunction with all three kinds of abilities in conjunction with target, and wherein (+)-KDT501 shows the higher affinity to all three kinds of targets.In described target, (+)-KDT501 with maximum affinity in conjunction with PPAR γ.Then evaluate (+)-KDT501 and the impact of (-)-KDT501 on PPAR activity.Two kinds of compounds all increase PPAR gamma activity and PPAR α or PPAR δ activity are had little or without impact.Yet, (+)-KDT501 on the impact of PPAR gamma activity, under all concentration, be (-)-KDT501 at least 3 of its impact times, show (+)-KDT501 is obviously more effective as PPAR γ activator.PPAR γ is main regulation thing in lipogenesis, glucose homeostasis and insulin sensitivity, and be the molecule target of thiazolidinedione, it makes cell to insulin sensitivity, and in liver, fatty tissue and skeletal muscle, has anti-diabetic effect (Tontonoz1994; Tontonoz1994).

The ability of the derivative induced lipid formation of KDT500, lipogenesis and PPAR gamma activity based on providing herein, provide one or more KDT500 derivants that provide or its method that pharmaceutical compositions of enantiomer-pure is induced lipid formation, induced lipolysis formation and/or activated PPAR γ substantially by drug treatment effective dose herein herein, and treatment there is the method for in this object needing, PPAR γ being modulated the disease responding.The example of the disease that modulation responds to PPAR γ comprises, for example, by improving glucose and the medicable disease of energy body inner equilibrium, include but not limited to type ii diabetes, obesity, hyperinsulinemia, metabolism syndrome, non-alcoholic fatty liver disease, nonalcoholic steatohepatitis, autoimmune disorder and proliferative disorders.

As mentioned above, former fixed PPAR gamma agonist has shown inflammation-inhibiting reaction.PPAR γ not only activates for the transcribing of the target gene of lipid metabolism, and lowers the expression (Pascual2005) of inflammation gene expression.Inflammatory reaction is subject to the inhibition of PPAR gamma agonist and diabetes and antiatherogenic effect closely related.In order to evaluate the effect of KDT501 in inflammation, (+)-KDT501 is evaluated to the ability that it suppresses expression various TNF-α-mediations, LPS-inflammatory factor that mediate and IL-1 β-mediation.Observed (+)-KDT501 suppresses TNF-α-mediation and expression LPS-mediation of being subject to of MMP-9, IL-1 β, MCP-1, RANTES and MIP-1 α in THP-1 cell, and PGE in inhibition rheumatoid arthritis synovial fluid fibroblast (RASF) cell ₂the expression that is subject to IL-1 β-mediation with MMP-13.In addition, observed (+)-KDT501 and (-)-KDT501 suppress DAPK1 activity, and wherein (+)-KDT501 demonstrates maximum usefulness.

KDT501 improves that insulin sensitivity and glucose regulate and the ability that lowers proinflammatory signal shows to affect the interactional mechanism between insulin signaling transduction and pathways of inflammation.A kind of potential mechanism of this activity is the stimulation by g protein coupled receptor.In order to evaluate this probability, the active impact of test (+)-KDT501 on the G-G-protein linked receptor GPR120 of omega-3 fatty acid sensitization.The activation of GPR120 improves insulin sensitivity and the (Oh2010 that reduces inflammation by suppressing NF-kB pathway; Talukdar2011).GPR120 has also shown to mediate the GLP-1 secretion (Hirasawa2005 in intestinal L cell; Hara2011) lipid and in increase adipose cell forms (Goth2007), all promotes diabetes effect.Observed (+)-KDT501 is with the EC of 30.3 μ M ₅₀be worth exciting GPR120 active, show (+)-KDT501 can partly activate its lipid of performance by GRP120 and form effect.

The ability of the derivative induced GPR120 activity of KDT500 based on providing herein, provide herein one or more KDT500 derivants that provide herein by drug treatment effective dose or its substantially the pharmaceutical compositions of enantiomer-pure treat the method for the disease that modulation responds to GPR120 in this object needing.In addition, the derivative induced GPR120 of KDT500 based on providing herein and PPAR gamma activity also suppress the ability of expression of the inflammatory factor of the various TNF-of being subject to α-mediations, LPS-mediation and IL-1 β-mediation, provide herein one or more KDT500 derivants that provide herein by drug treatment effective dose or its substantially the pharmaceutical compositions of enantiomer-pure suppress to have the method for the inflammation in the object of these needs.Also provide herein one or more KDT500 derivants that provide herein by drug treatment effective dose or its substantially the pharmaceutical compositions of enantiomer-pure treat various diseases relevant to inflammation in this object needing and/or inflammatory factor MMP-9, IL-1 β, MCP-1, RANTES, MIP-1 α, PGE ₂with one or more the method for disease of Horizontal correlation of rising in MMP-13.Such disease comprises, for example, atherosclerosis, atheromatous plaque unstability, autoimmune disease for example HIV/AIDS, nephropathy, tumor and diabetes of rheumatoid arthritis, cartilage degradation, osteoarthritis, allergic conditions, immune deficiency disorder for example.

As disclosed herein, in rat diabetes model, evaluate the ability that (+)-KDT501 changes glucose and lipid level.Under the dosage of 200mg/kg once a day, observe (+)-KDT501 significantly reduces glucose level.This reduction is far longer than to be observed in the rat with Or Metformin In Treating, and in rat with Mellitus Model With Pioglitazone, observe similar.Similarly, having observed (+)-KDT501 is reduced to and the almost identical degree of observing with pioglitazone triglyceride level.

In mouse model, twice administration every day (+)-KDT501 reduces circulation full blood glucose and insulin level effectively, in oral glucose tolerance test (OGTT), be reduced in glucose and Insulin area (AUC) under concentration-time curve, and the fat mass in obesity (DIO) mice of the induction that cuts down one's diet.In DIO mice, in 30 day time period, also observed the reduction trend of glycated hemoglobin (HbA1c) percentage ratio.

KDT500 derivant based on providing herein reduces the ability of circulating glucose, triglyceride and insulin level in rat and mouse model, supplying method herein, one or more KDT500 derivants that provide herein by drug treatment effective dose of the method or its substantially pharmaceutical compositions of enantiomer-pure are treated dysbolismus." dysbolismus " refers to any disease that causes homoiostasis to lose Metabolism control while using in this article.The example of dysbolismus comprises, for example, and diabetes, hyperglycemia, weightening finish, insulin resistant, dyslipidemia and hypercholesterolemia.In some embodiments, supplying method, the method by one or more KDT500 derivants that provide herein of drug treatment effective dose or substantially the pharmaceutical compositions of enantiomer-pure treat the diabetes in this object needing, comprise type ii diabetes.Similarly, supplying method in some embodiments, the method by one or more KDT500 derivants that provide herein of drug treatment effective dose or substantially the pharmaceutical compositions of enantiomer-pure reduce glucose and/or the lipid level having in this object needing.In some embodiments, the administration of described KDT500 derivant or compositions causes the reduction of blood sugar level and/or the reduction of triglyceride level.In some embodiments, the administration of described KDT500 derivant or compositions causes comprising one or more the reduction in other lipid levels of T-CHOL for example or LDL level.

In some embodiment of the method providing in this article, described to as if mammal, in some of these embodiments, described to as if people." have this need object " refers to suffer from after diagnosing object, the performance of modulation responds to PPAR γ disease or dysbolismus or shown modulation responds to PPAR γ disease or the object of one or more symptoms in dysbolismus, or based in heredity or environmental factors one or more and be considered to modulation responds to PPAR γ disease or the object of dysbolismus onset risk.

Term " treatment " while using in this article, with regard to disease, refer to prevention this disease, the outbreak that slows down this disease or tempo, minimizing suffer from risk, the prevention of this disease or postpone the symptom relevant to this disease development, alleviate or stop the symptom relevant with this disease, produce the disappearing wholly or in part of this disease, or its some combinations.

" the treatment effective dose " of KDT500 derivant or pharmaceutical compositions refers to the amount of the compositions of the curative effect that produces expectation in object while using in this article.Definite treatment effective dose is in specific object, with regard to curative effect, to produce this compound of the most effective result or the amount of compositions.This amount can be depending on the feature that many factors includes but not limited to this treatment compound and (comprises, for example, activity, pharmacokinetics, pharmacodynamics and bioavailability), physiology's situation of this object (comprises, for example, age, sex, disease type and stage, general condition, the reaction to given dose, and drug type), the character of the pharmaceutically acceptable carrier in said composition or carrier and route of administration and change.Technical staff in clinical and area of pharmacology can be by routine test, by monitoring target to taking the reaction of compound and correspondingly adjusting dosage and determine and treat effective dose.Extra guidance, referring to Remington:The Science and Practice of Pharmacy the 21st edition, Univ.of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005, its whole disclosing is incorporated herein by quoting.

In some embodiments, the KDT500 derivant providing herein or the treatment effective dose of compositions can be selected from every object approximately 10 every day ^-10g is to about 100g, approximately 10 ^-10g is to approximately 10 ^-3g, approximately 10 ^-9g is to approximately 10 ^-6g, approximately 10 ^-6g is to about 100g, about 0.001g to about 100g, about 0.01g to about 10g, or about 0.1g is to the active component (being KDT500 derivant) of about 1g.In some embodiments, the treatment effective dose of KDT500 derivant or compositions can calculate according to the body weight of object.For example, in some embodiments, treatment effective dose can be the about 100mg/kg of every object every day or more, about 150mg/kg or more, about 200mg/kg or more, about 250mg/kg or more, or about 300mg/kg or more active component (being KDT500 derivant).

In some embodiments, the compound providing herein or compositions can be once a day or are repeatedly carried out administration.In other embodiments, described compound or compositions can be less than and once carry out administration every day.For example, described compound or compositions can be once in a week, monthly once or per several months once carry out administration.The compound providing herein or the administration of compositions can be carried out in the time period in predetermined particular treatment, or can carry out indefinitely, or until reach specific treatment benchmark.For example, administration can be carried out until glucose and/or lipid level reach predetermined threshold value.In some embodiments, administration frequency can change in therapeutic process.For example, object can be accepted the administration of lower frequency in the process for the treatment of when reaching some treatment benchmark.

Compound disclosed herein or compositions can by any route of administration known in the art include but not limited to per os, spraying, through intestinal, per nasal, for example, through eye, parenteral or percutaneous (, topical cream agent or ointment, patch) to object administration." parenteral " refers to route of administration, it is general relevant with injection, comprise under socket of the eye, infusion, intra-arterial, capsule are interior, intracardiac, in Intradermal, intramuscular, intraperitoneal, lung, spinal column is interior, breastbone is interior, sheath is interior, under intrauterine, intravenous, arachnoidea, under capsule, subcutaneous, through mucous membrane or transtracheal administration.Preparation can parenteral the method for compositions be well known by persons skilled in the art or apparent, and at publication Remington:The Science and Practice of Pharmacy the 21st edition for example, Mack Publishing Company, Easton, describes in Pa. (2005) in more detail.Compositions can also be with the form administration of bolus, electuary or paste.

In some embodiments, provide medicine box, one or more KDT500 derivants that provide herein or its compositions of enantiomer-pure is substantially provided for it.In some embodiments, described medicine box provides operation instructions, for example dosage or the description of taking medicine.In some embodiments, described medicine box can be used to disease that in having this object needing treatment responds to PPAR γ or GPR120 modulation, treatment dysbolismus for example diabetes, reduce glucose and/or lipid level, inflammation-inhibiting, or the treatment disease relevant to inflammation.

The KDT500 derivative composition of the KDT500 derivant providing herein in some embodiments, and substantially enantiomer-pure can be used as flavoring agent.In some in these embodiments, described compound is as bitters.

Provide following examples to should not be construed as and limit the scope of the invention better the present invention to be described.The degree that concrete material is mentioned is only for purpose of explanation but not is intended to limit the present invention.Those skilled in the art can not exercise creative ability and develop without departing from the scope of the invention equivalent means or reaction reagent.In method described in should understanding in this article, can carry out many variations and still be within the scope of the present invention.The inventor is intended to such variation to comprise within the scope of the invention.

embodiment

embodiment 1: (crystallization and the sign of+) – and (-)-KDT500 derivant of enantiomer-pure substantially:

the crystallization of (+)-KDT501 (diagram I):

What 100g was obtained in Flos lupuli (Flos Humuli Lupuli) processing procedure is mainly that cis tetrahydrochysene-different alpha acid utilizes high-speed countercurrent chromatography (HSCCC) to carry out purification (Dahlberg2010) according to the method for having reported.HPLC and the analysis of UV peak area show that the product of gained is > 90%KDT500.Purified material for example, is reacted and changed into (+)-KDT501 by the potassium salt with 1 equivalent (KOH), and recrystallization is repeatedly to remove color.After reaching enough homogeneity, by the further recrystallization of (+)-KDT501 so that the crystallization of (+)-KDT501 has the size that is enough to be used in X-ray diffraction test.

Diagram I

Water (450 μ L) and (+)-KDT501 (49.5mg) are added in 4mL bottle, and with this bottle of sealing of lid, with heater (70 ° of C), heat until dissolve.Make this solution in 2 hours, be cooled to 35 ° of C.In at this moment, the scene of observing crystallization forms.This mixture is descended standing 8 hours in room temperature (22 ° of C), observe at this moment the formation of other crystallizations.Identification is suitable for the single crystallization of x-ray analysis, takes out carefully from solution, installs, and then carries out X-ray diffraction analysis.

the parsing of the crystalline texture of (+)-KDT501:

To, without color lump, be of a size of 0.37 * 0.30 * 0.28mm ³, be fixed on capillary glass tube together with oil.At Bruker APEX II Advances in crystal X-ray diffraction instrument, in Mo-radiation at-163 ℃ image data.For all groups, crystallization-to-detector distance is that 40mm and time of exposure are 10 seconds/degree.Sweep length is 0.5 °.Data acquisition 98.4% completes to 25 ° of θ.Amount to 68211 (merging) secondary reflection collected, the index covering: h=-26 to 28, k=-34 to 34, l=-16 to 14.16681 secondary reflections be symmetry independently and R _int=0.0386 shows that these data are good (average qualities 0.07).Indexing and cell parameter correction show former orthohomic lattice (primitive orthorhombic lattice).Observing space group is P2 ₁2 ₁2 (No.18).

Utilize APEX2 software kit (Bruker2007APEX2 (2.1-4 version), SAINT (7.34A version), SADABS (2007/4 edition), BrukerAXS Inc, Madison, WI) in SAINT, SADABS integration and drawing data in proportion.

According to the solution (SHELXS of direct method; SIR97 (Altomare J Appl Cryst32:115 (1999), Altomare J Appl Cryst26:343 (1993)) produces the complete heavy atom phasing model consistent with the structure of proposing.With SHELXL97 (Sheldrick, " SHELXL-97:Program for the Refinement of Crystal Structures, " University of Gottingen, Germany (1997); Mackay " MaXus:a computer program for the solution and refinement of crystal structures from diffraction data; " University of Glasgow, Scotland (1997)) by Fourier synthesis this structure that completes of difference.Dispersion factor is from Waasmair and Kirfel (Waasmaier Acta Crystallogr A51:416 (1995)).Hydrogen atom be placed in how much Utopian positions and the restrained parent that depends on them wherein C---H distance within the scope of 0.95-1.00 dust.Fixing isotropism thermal parameter U _eqthereby for CH, be the 1.2U of their parent Ueq _eqand in the situation of methyl, be their parent U _eq1.5U _eq.All non-hydrogen atoms are carried out to anisotropically precision processing by complete matrix method of least square.The crystalline texture of (+)-KDT501 is shown in Fig. 1.Crystallographic data is shown in Table 1, and three-dimensional atomic coordinates is shown in Table 2, and bond distance and angle are shown in Table 3, and anisotropy displacement parameter (anisotropy shift factor index different forms wherein :-2 π ²[h ²a* ²u ¹¹+ ...+2hka*b*U ¹²]) be shown in Table 4.

The asymmetric cell of (+)-KDT501 crystal form is by 4 (+)-KDT501 molecular composition with 4 potassium ion complexations.2 potassium ions and oxo bridge connect; Unordered hydrone and other two complexations.The ketone of organic moiety and potassium ion complexation produce tightly packed pattern.The asymmetric cell of (+)-KDT501 is shown in Fig. 2.Absolute configuration obtains (absolute structure parameter=0.04 (4)) from anomalous scattering.

the X-ray powder diffraction (XRPD) of (+)-KDT501 and heat are analyzed:

The Panalytical Xpert Pro diffractometer that use is furnished with Cu X-ray tube and Pixcel detector system carries out XRPD analysis.Isothermal sample is analyzed and is fixed between low density polyethylene films in transfer mode.XRPD diffraction pattern shows that (+)-KDT501 is crystallization (Fig. 3).

thermogravimetric analysis/differential thermal analyzer (TG/DTA):

Heat analysis is carried out on Perkin Elmer Diamond.Calibration criterion is indium and stannum.Sample is placed in to aluminum quality sample dish, inserts in TG stove and accurately weigh.Sample is heated from 300 ° of C of 30 ° of C – with 10 ° of speed of C/ minute in nitrogen current.Before the analysis of sample, make the hygral equilibrium of this stove at 30 ° of C.

TG/DTA thermogram (Fig. 4) demonstrates 1.37% loss of weight, shows to lose some solvents, is the indication of hydrate or solvate.Except the melting heat absorption when 145 ° of C start, do not have other incident heats to occur.

embodiment 2:(+) impact that-KDT501 forms lipid in 3T3-L1 adipose cell:

In 3T3-L1 adipose cell, evaluate the impact that (+)-KDT501 forms lipid.

Adipose cell (ATCC, Rockville, MD) before Mus 3T3-L1 is being supplemented with to 10% hyclone (FBS; ATCC) in DMEM (Invitrogen, Rockville, MD), cultivate.As front adipose cell, 3T3-L1 cell has fibroblastic outward appearance.They breed until they form and merge monolayer in culture fluid, and cell-cells contacting triggers G thereafter ₀/ G ₁growth retardation.After the follow-up insulin stimulating with 3-isobutyl group-1-methyl xanthane, dexamethasone and high dose impels cell to carry out for two days-and merge the asexual amplification of mitosis, leave cell cycle, and start to express adipose cell-specific gene.Differentiation induction after approximately 5 days, cell shows the adipose cell phenotype of characteristic lipid-filling.

By cell with approximately 1.5 * 10 ⁶the initial density of individual cell is seeded in 24-orifice plate and cultivates 2 days to merging.At the 0th day by (+)-KDT501(25,12.5,6.25 and 3.25 μ M for cell), rosiglitazone (10 μ M, positive control; Cayman Chemicals, Ann Harbor, MI) or DMSO(negative control) process.After this preliminary treatment, by adding by 10%FBS/DMEM (high glucose), 0.5mM methylisobutylxanthine (Sigma, St.Louis, MO), the division culture medium that forms of 0.5 μ M dexamethasone (Sigma) and 10 μ g/mL insulins (Sigma) makes cell differentiation lipoblast.After 2 days, this culture medium is replaced by the process culture medium (progression medium) being formed by 10 μ g/mL insulin/10%FBS/DMEM.In addition two days later, culture medium is replaced by the maintain base being formed by 10%FBS/DMEM.In the whole period of maturation (the 6th day/the 7th day), within every two days, with (+)-KDT501 or rosiglitazone/DMSO, process again cell.When adding new culture medium, also add new test material.

In whole test at the 0th day and within every two days, carry out microscopic evaluation with Adipocyte Differentiation lipoblast morphology before assessing.With Oil Red O dyeing quantization cell inner lipid.After the 8th day/the 9th day, discard carefully culture medium at not disturbance cellular layer in the situation that.10% formalin is added and hatch 15 minutes, with 60% isopropyl alcohol clean plate, and at room temperature dry 10 minutes.By the red O solution of the Oil of 300 μ L (0.36%/60% ethanol; Millipore, Billerica, MA) add and at room temperature hatch 10 minutes.With 70% ethanol clean plate, then water cleans 2 times.By adding 100% isopropyl alcohol of 200 μ L to extract dyestuff 20 minutes.150 μ L samples are transferred to 96 orifice plates and under 530nm, measure absorbance with plate reader (Thermo Electron Corp.).With 100% isopropyl alcohol as blank.

The results are summarized in Fig. 5.(+)-KDT501 mode with statistically significant under 6.25,12.5 and 25 μ M increases the lipid formation in 3T3-L1 adipose cell.The growth of observing can be compared with the growth of observing with rosiglitazone.

embodiment 3:(+)-KDT501 to TNF-α in THP-1 cell-and the inflammation of LPS-mediation because of the impact of son:

In person monocytic cell THP-1 cell, evaluate the antiphlogistic effects of (+)-KDT501.

According to manufacturer's description, THP-1 cell (ATCC, Manassas, VA) is cultivated in RPMI1640 under the existence of 10% serum.Cell use (+)-KDT501 is descended to preincubate 1 hour in different concentration (50,25,12.5 and 3.125 μ M), then use TNF-α (10ng/ml; Sigma, St.Louis, MO) or Escherichia coli LPS (1 μ g/ml; Sigma, St.Louis, MO) (16-20 hour) stimulation of spending the night.With ELISA test kit (GE Healthcare, Piscataway, NJ), measure the MMP-9 level in culture medium, and use Milliplex MAP human cell factor/chemotactic factor test kit (Millipore, Billerica, MA) to measure cytokine.Use Luminex100 ^tMiS quantifying analytes.By 5-parameter logarithm methods analyst data.

The results are shown in Fig. 6-10.(+)-KDT501 suppresses the expression being caused by TNF-α and LPS-of MMP-9 (Fig. 6), IL-1 β (Fig. 7), MCP-1 (Fig. 8), RANTES (Fig. 9) and MIP-1 α (Figure 10) with dose-dependent manner.

embodiment 4:(+) impact of-KDT501 on the inflammatory factor of IL-1 β-mediation in RASF:

In rheumatoid arthritis synovial fluid fibroblast (RASF), evaluate the antiphlogistic effects of (+)-KDT501.

People RASF cell (Asterand, Detroit, MI) is cultivated and maintained in DMEM/F12 (1:1) culture medium under 10% hyclone exists.By cell in 24-orifice plate with 1x10 ⁴the density of individual cells/well time culture also makes it in 2 days, reach 70-80% fusion.The cell merging under the ultimate density of 0.1%DMSO in the culture medium of serum-free is hatched 1 hour under different concentration (50,25,12.5 and 6.25 μ M) with (+)-KDT501, then use IL-1 β (10ng/ml) to stimulate 20-24 hour.Use ELISA test kit (GE Healthcare, Piscataway, NJ) to measure the MMP-13 level in culture medium, and measure PGE with immunity detection reagent (Assay Designs, Ann Arbor, MI) ₂level.

The results are shown in Figure 11 and 12.(+)-KDT501 suppresses the PGE of IL-1 β-induction ₂(Figure 11) and the expression of MMP-13 (Figure 12).

the competitive binding of embodiment 5:KDT501 to PPAR γ, SCN2A and AGTR2:

(+)-KDT501 evaluates respectively the receptor y of peroxisome proliferation body-activation (PPAR γ), valtage-gated sodium channel II type (SCN2A) and the competitive binding of blood angiotonin II receptor I I type (AGTR2) under the existence of agonist rosiglitazone, veratridine and AngiotensionⅡ.

For PPAR γ, by cell membrane homogenate (8 μ g protein) under 4 ° of C with 5nM[ ³h] rosiglitazone have or without the buffer agent that comprises 10mM Tris-HCl (pH8.2), 50mM KCl and 1mM DTT of (+)-KDT501, exist under hatch 120 minutes, and measure non-specific binding under 10 μ M rosiglitazones exist.

For SCN2A, by corticocerebral cell membrane homogenate (200 μ g protein) under 22 ° of C with 10nM[ ³h] batrachotoxin having or without comprising 50mMHepes/Tris (pH7.4), 130mM choline chloride, 5.4mM KCl, 0.8mM MgSO under (+)-KDT501 ₄, 1g/l glucose, 0.075g/l scorpion venom and 0.1%BSA buffer agent in hatch 60 minutes, and measure non-specific binding under the existence of 300 μ M veratridines.

For AGTR2, by cell membrane homogenate (5 μ g protein) under 37 ° of C with 0.01nM[ ¹²⁵i] CGP42112A (AngiotensionⅡ receptor II type agonist) having or without comprising 50mM Tris-HCl (pH7.4), 5mM MgCl under (+)-KDT501 ₂, 1mM EDTA and 0.1%BSA buffer agent in hatch 240min, and measure non-specific binding under the existence of 1 μ M Angiotensin II.

After hatching, sample is passed through to the glass fiber filter (GF/B with 0.3%PEI pre-preg, Packard) fast filtering use 50mM Tris-HCl ice-cold for 96-sample cell catcher (Unifilter, Packard) to rinse for several times under vacuum.Filter is dry, then use flicker intermixture (Microscint0, Packard) in scintillation counter (Topcount, Packard) to radiocounting.

The results are summarized in table 5.(+)-KDT501 shows under target agonist ligand exists in conjunction with the ability of all three kinds of targets, wherein the highest with the binding affinity of PPAR γ.

Table 5:

Using (-)-KDT501 to substitute (+)-KDT501 repeats as mentioned above in conjunction with test.These the results are summarized in table 6.Be similar to (+)-KDT501, (-)-KDT501 shows under target agonist ligand exists the ability in conjunction with all three kinds of targets.Yet, the IC of (-)-KDT501 to all three kinds of targets ₅₀and K _ihigher than (+)-KDT501 is shown, go out.

Table 6:

the impact of embodiment 6:KDT501 on PPAR activity:

Utilize PPAR reporter gene assay (INDIGO Biosciences, PA) to evaluate the functional impact of (+)-KDT501 to PPAR α, PPAR δ and PPAR gamma activity.This mensuration is utilized non-human mammal cell, this cell by genetically engineered the composition high level expression of PPAR α, PPAR δ or PPAR γ to be provided and to comprise the specific luciferase report gene to suitable PPAR.By agonist in conjunction with after activating, the expression of PPAR induction luciferase report gene.Therefore luciferase activity provides the succedaneum of PPAR activity in the cell of mensuration agonist-processing.

Report cell is seeded on 96-orifice plate with 100 μ L/ holes, and 100 μ L (+)-KDT501 is added in each hole with different ultimate density (25,12.5,6.25,3.125,1.56 and 0.78 μ M).For PPAR γ test, rosiglitazone (1,0.5,0.25,0.125,0.063 and 0.031 μ M) and telmisartan (10,5,2.5,1.25 and 0.625 μ M) are as positive control.For PPAR α test, GW590735 (10,5,1,0.5,0.25,0.125,0.063 and 0.031 μ M) is as positive control, and rosiglitazone (1,0.5,0.25,0.125,0.063 and 0.031 μ M) is as negative control.For PPAR δ test, GW0742 (1,0.5,0.25,0.125,0.0625,0.031,0.016 and 0.008 μ M) is as positive control, and rosiglitazone (1,0.5,0.25,0.125,0.063 and 0.031 μ M) is as negative control.For every kind of test, 0.1%DMSO is also as negative control.By plate in moistening incubator at 37 ° of C and 5%CO ₂lower incubation 20 hours.After incubation, discard cell culture medium, and process cell 15 minutes with the luciferase detection agent of 100 μ L.Use illumination meter (Victor2, Perkin Elmer) analysis plates.Utilize GraphPad Prism to analyze average relative light unit (RLU) and standard deviation.

In PPAR γ test, positive agonist contrast (rosiglitazone) increases PPAR gamma activity as expected.Telmisartan, is a kind of known portions agonist of PPAR γ, also increases PPAR gamma activity.(+)-KDT501 increases PPAR gamma activity in significant mode statistically, and as part PPAR gamma excitomotor activity consistent (Figure 13).(+)-KDT501 has little or without impact (Figure 14 and 15) on PPAR α and PPAR δ activity.

Use (+)-KDT501 (12.5,6.25,3.125,1.56 and 0.78 μ M) and (-)-KDT501 (25,12.5,6.25,3.125,1.56 and 0.78 μ M), repeat as mentioned above PPAR activity test.(-)-KDT501 increases PPAR gamma activity, but with the degree significantly less than (+)-KDT501 (Figure 16).To the results are summarized in table 7 of two kinds of compounds.

Table 7:

embodiment 7:(+) impact of-KDT501 on GPR120 reporter gene activity:

(+)-KDT501 is the CHO-K1/GPR120/G that utilizes stably express GPR120 on the impact of GPR120 activity _{α 15}cell is evaluated.GPR120 activity is to be reacted and measured by intracellular Ca2+.

By CHO-K1/GPR120/G _{α 15}cell goes down to posterity the cell health that remains best regularly, and cultivates in Ham ' the s F12 that is supplemented with 10% hyclone, 200 μ g/mL bleomycin (zeocin) and 100 μ g/mL hygromycin.Before 18 hours on-tests, on the plate of the black wall clear bottom in 384-hole, by cell, the density with 20,000 cells/well is seeded in the growth medium of 20 μ L, and maintains 37 ° of C/5%CO ₂under.Cell is by load calcium-4, and uses fluorescent imaging plate reader (FLIPR) from 1 second to 20 seconds, to obtain baseline fluorescence reading.At the 20th second, (+)-KDT501 (5x ultimate density) was injected towards the flat board at reading, and monitored fluorescence signal 100 seconds (the 21st second to the 120th second).Alpha-linolenic acid (LA), eicosapentaenoic acid (EPA) and docosahexenoic acid (DHA) are with comparing.

Use ScreenWorks (3.1 editions) carry out data acquisition and analysis and export Excel to.For first 20 seconds, mean fluorecence value was calculated as baseline reading.Maximum fluorescence unit for intensity level (21s to 120s) of relative fluorescence unit (Δ RFU) deducts baseline reading and calculates.The data analysis guide of being write by GenScript is used for analyzing EC ₅₀.Use GraphPad Prism4 tetra-parameter logarithmic equation Y=Bottom+ (Top-Bottom)/(1+10^ ((LogIC ₅₀-X) * the HillSlope)) dose-effect curve of matching antagonist, wherein X is the logarithm of concentration, Y is reaction.

(+)-KDT501 shows GPR120 agonist activity and has the EC of 30.3 μ M ₅₀value (Figure 17).The EC of α-LA, DHA and EPA ₅₀value is respectively 36.1 μ M, 35.3 μ M and 116.9 μ M.

the impact of embodiment 8:KDT501 on DAPK1 activity:

(+)-KDT501 and (-)-KDT501 have been evaluated to the active impact of death-associated protein kinase 1 (DAPK1).

By people DAPK1 and 8mM MOPS, pH7.0,0.2mM EDTA, 250 μ M ZIP peptide substrates (KKLNRTLSFAEPG), 10mM MgOAc and [γ- ³³p-ATP] (the about 500cpm/pmol of activity specific, required concentration) incubation together.(+)-KDT501 or (-)-KDT501 are added in reactant mixture with different concentration (100,30,10,1,0.3,0.1,0.03,0.01 μ M).By adding the initiation reaction of MgATP mixture.At room temperature incubation is after 40 minutes, by adding 3% phosphoric acid solution cessation reaction.10 μ L reactant mixture points, on P30 filter pad, are cleaned 3 times 5 minutes and first in methanol wash once, are then dried, the line flicker of going forward side by side counting in 75mM phosphoric acid.

The results are shown in Figure 18.(+)-KDT501 and (-)-KDT501 suppress DAPK1 activity in dosage-dependent mode.IC to (+)-KDT501 and (-)-KDT501 calculating ₅₀value is respectively 2.65 μ M and 40.9 μ M, shows to differ approximately 15 times between this two compound.These data show that (+)-KDT501 is more effective than (-)-KDT501 aspect inhibition DAPK1 activity.

embodiment 9:(+)-KDT501 in rat diabetes model to glucose and triglyceride water flat impact:

By male Zucker obesity indiabetes in six week age (ZDF) the rat randomization of 64 glucose levels with 175-300mg/dL and be divided into 8 administration groups: 1) carrier (0.5% methylcellulose (w/v), 0.2%Tween80 (w/v) only; Negative control), 2) (+)-KDT50125mg/kg, 3) (+)-KDT50150mg/kg, 4) (+)-KDT501100mg/kg, 5) (+)-KDT501200mg/kg, 6) metformin 200mg/kg (positive control), 7) metformin 200mg/kg and (+)-KDT501100mg/kg, and 8) pioglitazone 30mg/kg (positive control).By medicine twice oral administration every day 33 days.

When randomization, animal is weighed, weigh once in a week thereafter, and the measured body weight value based on nearest is calculated dosage.Before randomization 3 days, when randomization, and after begin treatment the 15th day and the 29th day, by afterbody, get blood and gather blood sample.These blood samples are used for utilizing blood glucose meter to carry out full blood glucose evaluation, and are used for measuring blood triglyceride level.Within the 2nd day and the 29th day after begin treatment, by qNMR, test health and form.Within the 31st day and the 32nd day after begin treatment, carry out oral glucose tolerance test (OGTT).Rat carried out overnight fast before OGTT, and the 15th, 30,60,90 and 120 minutes before glucose pill and after glucose pill get blood for glucose and insulin carry out afterbody.Glucose pill is 2g glucose/kg body weight.At the 33rd day, for analyzing, PK carries out cardiac puncture.

Full blood glucose the results are shown in Figure 19.As expected, negative control rat shows the remarkable and stable growth of full blood glucose sugar level in research process.The positive control rat of accepting pioglitazone shows glucose level and significantly declines, and the positive control rat of accepting metformin shows glucose level, declines a little.With 25,50 or the dosage of the 100mg/kg rat of accepting (+)-KDT501 show with negative control rat in those almost identical glucose levels of observing.Yet the remarkable reduction of accepting the glucose level that the rat of (+)-KDT501 shows with the dosage of 200mg/kg is larger more than what observe in metformin control rats, and almost with in pioglitazone control rats, observe identical.

Triglyceride level the results are shown in Figure 20.The rat of accepting metformin is compared with negative control rat at the 15th day and the 29th day and shows the growth of triglyceride level, and the rat of accepting pioglitazone shows significant reduction.With 25,50 or the dosage of the 100mg/kg rat of accepting (+)-KDT501 show and those similar triglyceride level of observing in negative control rat.Yet, with the dosage of 200mg/kg, accept the remarkable reduction that the rat of (+)-KDT501 shows triglyceride level, almost with in pioglitazone control rats, observe equally large.

embodiment 10:(+)-KDT501 is in obesity (DIO) mouse model of high fat diet induction impact on glucose, insulin, HbA1 C (HbA1C) and fat mass:

By the male mice randomization in 14 week age and be divided into 7 administration groups, every group has 16 animals: 1) carrier (0.5% methylcellulose (w/v), 0.2%Tween80 (w/v) only; Negative control), 2) (+)-KDT50125mg/kg, 3) (+)-KDT50150mg/kg, 4) (+)-KDT501100mg/kg, 5) (+)-KDT501200mg/kg, 6) metformin 200mg/kg (positive control), and 7) pioglitazone 30mg/kg (positive control).By medicine twice oral administration every day 30 days.

When randomization, animal is weighed, weigh once in a week thereafter, and the measured body weight value based on nearest is calculated dosage.With high fat diet (40%Kcal) (TD95217; By Covance Laboratories, Greenfield, IN preparation) raising mice.Within the-5 days and the 28th day after begin treatment, by qNMR, test health and form.Within the 29th day after begin treatment, gather blood, and measure HbA1 C (HbA1C) level.Within the 30th day after begin treatment, carry out oral glucose tolerance test (OGTT).Before OGTT, mice carries out overnight fast, and the 15th, 30,60,90 and 120 minutes before glucose pill and after glucose pill get blood for glucose and insulin carry out afterbody.Glucose pill is 2g glucose/kg body weight.OGTT result is evaluated by the area under curve (AUC) measured value and Dunnett check.

Blood-glucose from OGTT the results are shown in Figure 21.The positive control mice of accepting pioglitazone or metformin shows the remarkable reduction of glucose level.Similarly, with 100 or the dosage of 200mg/kg accept the remarkable reduction that the mice of (+)-KDT501 shows glucose level.

Insulin from OGTT the results are shown in Figure 22.The positive control mice of accepting pioglitazone or metformin shows the remarkable reduction of insulin level.Similarly, with the dosage of 200mg/kg, accept the remarkable reduction that the mice of (+)-KDT501 shows insulin level.

HbA1C the results are shown in Figure 23.Between the negative control of carrier only and positive control metformin administration group, observe the significant difference of HbA1C.The administration of (+)-KDT501 produces the trend that trend HbA1C suppresses.

Fat mass the results are shown in Figure 24.The positive control mice of accepting metformin shows the minimizing of fat mass, and the positive control mice of accepting pioglitazone does not show difference.With 100 or 200mg/kg administration (+)-KDT501 compare the significant difference that produces fat mass with negative control mice.

embodiment 11: by .beta.-bitter acid asymmetric synthesis (+)-KDT500 or (-)-KDT500:

Diagram II

Step 1: by .beta.-bitter acid (II) tetrahydrobiopterin synthesis deoxidation .alpha.-bitter acid (VI).

10% solution (w/v) of preparation .beta.-bitter acid in methanol.The dense HCl (aq) and the 10%Pd/C that add catalytic amount.In the lower mixture that stirs gained of hydrogen (1.1atm) 3 hours, and monitor reaction by HPLC.After reaction completes, by removing by filter catalyst, and concentrated filtrate obtains tetrahydrochysene deoxidation .alpha.-bitter acid (VI) in a vacuum, and it can be without being further purified for next reaction.

Step 2A: by tetrahydrochysene deoxidation .alpha.-bitter acid (VI) asymmetric synthesis (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI).

Possibility 2A (i): by tetrahydrochysene deoxidation .alpha.-bitter acid (VI) asymmetric synthesis (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI).

Preparation tetrahydrochysene deoxidation .alpha.-bitter acid (VI) 14% (w/v) solution in anhydrous THF is also cooled to-78 ° of C with acetone/the dry ice bath.Diisopropylethylamine (DIEA) is (1.2eq) cooling and drop to the solution of (VI).This solution is transferred to (the CH by 2.2eq Cu by sleeve pipe ₃cN) ₄pF ₆[(-)-eulexine] obtaining under-78 ° of C under argon with two amine ligands of 2.3eq ₂-Cu ₂-O ₂complex) in/THF.The mixture of gained is stirred 16 hours at the temperature of-78 ° of C or expectation.By the moisture sulphuric acid cessation reaction of 5% (by weight) of 4 volumes.With ethyl acetate (* 3) extraction mixture, and the extract merging with 5% sulphuric acid, water and salt water washing, by dried over sodium sulfate, then concentrate in a vacuum (Dong2008).

Possibility 2A (ii): by tetrahydrochysene deoxidation .alpha.-bitter acid (VI) asymmetric synthesis (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI).

According to United States Patent (USP) 7,704,715 and wherein described in contained document, build and purification can change into tetrahydrochysene deoxidation .alpha.-bitter acid (VI) the applicable P450 mutant enzyme of (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI).The blood red prime field enzyme of P450 (heme domain enzyme) and 1-2mM tetrahydrochysene deoxidation .alpha.-bitter acid (VI)/500 μ L100mM tris-HCI that typical reaction comprises 1-4 μ M purification, pH8.2.To merge below: 713 μ L pure water, 200 μ L0.100M tris-HCl pH8.2,100mM ultimate density, 2 μ L tetrahydrochysene deoxidation .alpha.-bitter acid (VI)/DMSO (1mM ultimate density).

This reaction is by adding 1-10mM H ₂o ₂cause, and monitor maximum conversion rate by HPLC.This reaction is by adding 7.5 μ L6M HCl to stop.(-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI) obtains by extraction post processing, and carries out purification as previously mentioned.

Optional 2A (iii): from tetrahydrochysene deoxidation .alpha.-bitter acid (VI) asymmetric synthesis (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI).

According to U.S. Patent Publication 2010/0144547 and wherein contained document produce and tetrahydrochysene deoxidation .alpha.-bitter acid (VI) can be changed into the microorganism of (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI).According to following fermentation condition, under the existence of tetrahydrochysene deoxidation .alpha.-bitter acid (VI), cultivate this microorganism: there is the 500mL culture containing the LB culture medium of 30mg/L kanamycin.OD 1.0 ₆₀₀under, by the final OD of cell concentration to 5.0 ₆₀₀and induce with 1mM IPTG.Add tetrahydrochysene deoxidation .alpha.-bitter acid (VI) to the ultimate density of 1mM and 4mM.At different time points, gather culture samples, centrifugal, filter, and it is upper to be injected at HPLC (5 μ L), then measures maximum conversion rate.After maximum conversion, fluid medium is acidified to pH2.0, be extracted with ethyl acetate, dry, and be dissolved in hexane again.By extraction post processing, obtain (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI), and carry out as previously mentioned purification.

Various fermentation medium are LB well known in the art, F1 or TB fermentation medium can be used for or through adapting to for embodiments of the present invention disclosed herein, comprise LB, TB and F1 culture medium for example.Through adapt to be used for cultivating organism for example other culture medium of aspergillus terreus (A.terreus) and feathering Monascus anka Nakazawa et sato (M.pilosus) be also well known in the art (for example, referring to, Miyake2006; Hajjaj2001).

Step 2B: from tetrahydrochysene deoxidation .alpha.-bitter acid (VI) asymmetric synthesis (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI).

Optional 2B (i): from tetrahydrochysene deoxidation .alpha.-bitter acid (VI) asymmetric synthesis (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI).

Preparation tetrahydrochysene deoxidation .alpha.-bitter acid (VI) 14% (w/v) solution in anhydrous THF, and be cooled to-78 ° of C with acetone/the dry ice bath.Diisopropylethylamine (DIEA) is (1.2eq) cooling and drop in the solution of (VI).This solution is transferred to (the CH by 2.2eq Cu by sleeve pipe ₃cN) ₄pF ₆[(+)-eulexine] obtaining at-78 ° of C under argon with 2.3eq bis-amine ligands ₂-Cu ₂-O ₂complex) in/THF.The mixture of gained is stirred 16 hours at the temperature of-78 ° of C or expectation.By the moisture sulphuric acid cessation reaction of 5% (by weight) of 4 volumes.Be extracted with ethyl acetate this mixture (* 3), and the extract merging with 5% sulphuric acid, water and salt water washing, by dried over sodium sulfate, then concentrate in a vacuum (Dong2008).

Optional 2B (ii): from tetrahydrochysene deoxidation .alpha.-bitter acid (VI) asymmetric synthesis (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI).

According to United States Patent (USP) 7,704,715 and wherein described in contained document, build and purification can change into tetrahydrochysene deoxidation .alpha.-bitter acid (VI) the P450 mutant enzyme of (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI).Typical reaction is included in 500 μ L100mM Tris-HCI, the blood red prime field enzyme of P450 and the 1-2mM tetrahydrochysene deoxidation .alpha.-bitter acid (VI) of 1-4 μ M purification in pH8.2.To merge below: 713 μ L purified water, 200 μ L0.100M tris-HCl pH8.2,100mM ultimate density, 2 μ L tetrahydrochysene deoxidation .alpha.-bitter acid (VI)/DMSO (1mM ultimate density).

By adding 1-10mM H ₂o ₂initiation reaction, and monitor maximum conversion rate by HPLC.By adding 7.5 μ L6M HCI cessation reactions.By extraction post processing, obtain (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI), and carry out as previously mentioned purification.

Optional 2B (iii): from tetrahydrochysene deoxidation .alpha.-bitter acid (VI) asymmetric synthesis (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI).

According to U.S. Patent Publication 2010/0144547 and wherein contained document produce and tetrahydrochysene deoxidation .alpha.-bitter acid (VI) can be changed into the microorganism of (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI).According to following fermentation condition cultivating microorganism under the existence of tetrahydrochysene deoxidation .alpha.-bitter acid (VI): there is the 500mL culture containing the LB culture medium of 30mg/L kanamycin.OD 1.0 ₆₀₀under, by the final OD of cell concentration to 5.0 ₆₀₀and induce with 1mM IPTG.Add tetrahydrochysene deoxidation .alpha.-bitter acid (VI) to the ultimate density of 1mM and 4mM.At different time points, gather culture samples, centrifugal, filter, and be injected at HPLC upper (5 μ L), then measure maximum conversion rate.After maximum conversion, fluid medium is acidified to pH2.0, be extracted with ethyl acetate, dry, and be dissolved in hexane again.By extraction post processing, obtain (-)-tetrahydrochysene .alpha.-bitter acid, and carry out as previously mentioned purification.

Various fermentation medium are LB well known in the art, F1 or TB fermentation medium can be used for or through adapting to for embodiments of the present invention disclosed herein, comprise LB, TB and F1 culture medium for example.Through adapt to for cultivate organism for example other culture medium of aspergillus terreus and feathering Monascus anka Nakazawa et sato be also well known in the art (for example, referring to, Miyake2006; Hajjaj2001).

Step 3A: from (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI) asymmetric synthesis (+)-KDT500.

Preparation (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI) 50% (w/v) solution in water, and in the container of sealing, be warming up to 85 ° of C and stir.By MgSO ₄(0.6eq) in 5 minutes, be added to this solution continuous stirring.In the container of sealing, under agitation drip KOH solution (38.25% (w/v), 1.0eq).Make reaction temperature then in 3 hours or until remain on 85 ° of C while judging that by HPLC isomerization completes.By by the H of the water of the isopropyl alcohol of 2 volumes, 2 volumes and 1 equivalent ₂sO ₄be added to reactant mixture and form the free acid of (+)-KDT-500.This mixture is mixed until complete and homogeneous is now carried out extractive reaction mixture (3x) with the dichloromethane of 2 volumes.Organic extract liquid is concentrated in a vacuum, then be dissolved in hexane, Na used ₂sO ₄dry, filter, concentrated in a vacuum, and under 0.070mbar dried overnight.End-product is the free acid form of (+) KDT-500.Be further purified and can be undertaken by epimerization, chromatography, complexation and/or crystallization.

Step 3B: by (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI) asymmetric synthesis (-)-KDT500.

Preparation (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI) 50% (w/v) solution in water, and be under agitation warming up to 85 ° of C in the container of sealing.By MgSO ₄(0.6eq) under continuous stirring, in 5 minutes, be added in solution.To sealing container in, under agitation drip KOH solution (38.25% (w/v), 1.0eq).Make reaction temperature then in 3 hours or until remain on 85 ° of C when HPLC judgement isomerization completes.By by the H of the water of the isopropyl alcohol of 2 volumes, 2 volumes and 1 equivalent ₂sO ₄be added to reactant mixture and form the free acid of (-)-KDT-500.This mixture is mixed until complete and homogeneous is now carried out extractive reaction mixture (3x) with the dichloromethane of 2 volumes.Organic extract liquid is concentrated in a vacuum, then be dissolved in hexane, Na used ₂sO ₄dry, filter, concentrated in a vacuum, and under 0.070mbar dried overnight.End-product is the free acid form of (-) KDT-500.Be further purified and can be undertaken by epimerization, chromatography, complexation and/or crystallization.

embodiment 12: by deoxidation .alpha.-bitter acid asymmetric synthesis (+)-KDT500 or (-)-KDT500:

Diagram III

Step 1A: by deoxidation .alpha.-bitter acid (I) asymmetric synthesis (-)-.alpha.-bitter acid ((-)-IV).

Possibility 1A (i): by deoxidation .alpha.-bitter acid (I) asymmetric synthesis (-)-.alpha.-bitter acid ((-)-IV).

Preparation deoxidation .alpha.-bitter acid (I) 14% (w/v) solution in anhydrous THF is also cooled to-78 ° of C with acetone/the dry ice bath.Diisopropylethylamine (DIEA) is (1.2eq) cooling and drop in the solution of (VI).This solution is transferred to (the CH by 2.2eq Cu by sleeve pipe ₃cN) ₄pF ₆[(-)-eulexine] obtaining under-78 ° of C under argon with two amine ligands of 2.3eq ₂-Cu ₂-O ₂complex) in/THF.The mixture of gained is stirred 16 hours at the temperature of-78 ° of C or expectation.By the moisture sulphuric acid cessation reaction of 5% (by weight) of 4 volumes.By ethyl acetate (* 3), extract this mixture, and the extract merging with 5% sulphuric acid, water and salt water washing, by dried over sodium sulfate, then concentrate in a vacuum (Dong2008).

Possibility 1A (ii): by deoxidation .alpha.-bitter acid (I) asymmetric synthesis (-)-.alpha.-bitter acid ((-)-IV).

According to United States Patent (USP) 7,704,715 and wherein described in contained document, build and purification can change into deoxidation .alpha.-bitter acid (I) the P450 mutant enzyme of (-)-.alpha.-bitter acid ((-)-IV).The blood red prime field enzyme of P450 and 1-2mM .alpha.-bitter acid (I)/500 μ L100mM tris-HCl that typical reaction comprises 1-4 μ M purification, pH8.2.To merge below: 713 μ L purified water, 200 μ L0.100M tris-HCl pH8.2,100mM ultimate density, 2 μ L deoxidation .alpha.-bitter acid (I)/DMSO (1mM ultimate density).

By adding 1-10mM H ₂o ₂initiation reaction is also monitored maximum conversion rate by HPLC.By adding 7.5 μ L6M HCl cessation reactions.By extraction post processing, obtain (-)-.alpha.-bitter acid ((-)-IV), and carry out as previously mentioned purification.

Possibility 1A (iii): by deoxidation .alpha.-bitter acid (I) asymmetric synthesis (-)-.alpha.-bitter acid ((-)-IV).

According to U.S. Patent Publication 2010/0144547 and wherein contained document produce and .alpha.-bitter acid (I) can be changed into the microorganism of (-)-.alpha.-bitter acid ((-)-IV).Then according to following fermentation condition, under existing, deoxidation .alpha.-bitter acid (I) cultivates this microorganism: there is the 500mL culture containing the LB culture medium of 30mg/L kanamycin.OD 1.0 ₆₀₀under, by the final OD of cell concentration to 5.0 ₆₀₀and induce with 1mM IPTG.Add deoxidation .alpha.-bitter acid (I) to the ultimate density of 1mM and 4mM.At different time points, gather culture samples, centrifugal, filter, and it is upper to be injected at HPLC (5 μ L), then measures maximum conversion rate.After maximum conversion, fluid medium is acidified to pH2.0, be extracted with ethyl acetate, dry, and be dissolved in hexane again.By extraction post processing, obtain (-)-.alpha.-bitter acid ((-)-IV), and carry out as previously mentioned purification.

Step 1B: by deoxidation .alpha.-bitter acid (I) asymmetric synthesis (+)-.alpha.-bitter acid ((+)-IV).

Possibility 1B (i): by deoxidation .alpha.-bitter acid (I) asymmetric synthesis (+)-.alpha.-bitter acid ((+)-IV).

Preparation deoxidation .alpha.-bitter acid (I) 14% (w/v) solution in anhydrous THF is also cooled to-78 ° of C with acetone/the dry ice bath.Diisopropylethylamine (DIEA) is (1.2eq) cooling and drop in the solution of (VI).This solution is transferred to (the CH by 2.2eq Cu by sleeve pipe ₃cN) ₄pF ₆[(+)-eulexine] 2-Cu obtaining under-78 ° of C under argon with two amine ligands of 2.3eq ₂-O ₂complex) in/THF.The mixture of gained is stirred 16 hours at the temperature of-78 ° of C or expectation.By the moisture sulphuric acid cessation reaction of 5% (by weight) of 4 volumes.By ethyl acetate (* 3), extract this mixture, and the extract merging with 5% sulphuric acid, water and salt water washing, by dried over sodium sulfate, then concentrate in a vacuum (Dong2008).

Optional 1B (ii): from deoxidation .alpha.-bitter acid (I) asymmetric synthesis (+)-.alpha.-bitter acid ((+)-IV).

According to United States Patent (USP) 7,704,715 and wherein described in contained document, build and purification can change into deoxidation .alpha.-bitter acid (I) the P450 mutant enzyme of (+)-.alpha.-bitter acid ((+)-IV).Typical reaction is included in 500 μ L100mM tris-HCl, the blood red prime field enzyme of P450 and the 1-2mM .alpha.-bitter acid (I) of 1-4 μ M purification in pH8.2.To merge below: 713 μ L purified water, 200 μ L0.100M tris-HCl pH8.2,100mM ultimate density, 2 μ L deoxidation .alpha.-bitter acid (I)/DMSO (1mM ultimate density).

By adding 1-10mM H ₂o ₂initiation reaction, and monitor maximum conversion rate by HPLC.By adding 7.5 μ L6M HCI cessation reactions.By extraction post processing, obtain (+)-.alpha.-bitter acid ((+)-IV), and carry out as previously mentioned purification.

Optional 1B (iii): from deoxidation .alpha.-bitter acid (I) asymmetric synthesis (+)-.alpha.-bitter acid ((+)-IV).

According to U.S. Patent Publication 2010/0144547 and wherein contained document produce and deoxidation .alpha.-bitter acid (I) can be changed into the microorganism of (+)-.alpha.-bitter acid ((+)-IV).Then according to following fermentation condition cultivating microorganism under the existence of (+)-.alpha.-bitter acid ((+)-IV): there is the 500mL culture containing the LB culture medium of 30mg/L kanamycin.OD 1.0 ₆₀₀under, by the final OD of cell concentration to 5.0 ₆₀₀and induce with 1mM IPTG.Add deoxidation .alpha.-bitter acid (I) to the ultimate density of 1mM and 4mM.At different time points, gather culture samples, centrifugal, filter, and be injected at HPLC upper (5 μ L), then measure maximum conversion rate.After maximum conversion, fluid medium is acidified to pH2.0, be extracted with ethyl acetate, dry, and be dissolved in hexane again.By extraction post processing, obtain (+)-.alpha.-bitter acid ((+)-IV), and carry out as previously mentioned purification.

Step 2A: from (-)-.alpha.-bitter acid ((-)-IV) asymmetric synthesis (+)-cis IsoHopCO2N. ((+)-VIII) (RTP:K008-49)

By 98.9mg (-)-.alpha.-bitter acid ((-)-IV) with 170 μ L water dilutions and be under agitation warming up to 85 ° of C in the 4mL bottle of adding a cover.The MgSO that adds 19.8mg (0.6eq) ₄, and continue to stir 5 minutes.38.25% (w/v) KOH solution of 31 μ L (1.03eq) is added in this solution, and makes reaction proceed 3 hours under 85 ° of C, now through HPLC judgement, reacted.The product of gained is used for producing free acid or the Mg salt of (+)-cis IsoHopCO2N. ((+)-VIII).

By by 0.5mL isopropyl alcohol, 0.5mL water and 1 equivalent H ₂sO ₄be added to reactant mixture and form the free acid of (+)-cis IsoHopCO2N. ((+)-VIII).This mixture is mixed until complete and homogeneous is now carried out extractive reaction mixture 3 times with 0.5mL dichloromethane.Organic extract liquid is concentrated in a vacuum, then be dissolved in hexane, Na used ₂sO ₄dry, filter, and in a vacuum concentrated and under 0.070mbar dried overnight, the free acid form of obtain (+)-cis IsoHopCO2N. ((+)-VIII).

By reactant mixture is removed and is under agitation added 0.5mL ethyl acetate until dissolve the Mg salt that forms (+)-cis IsoHopCO2N. ((+)-VIII) completely from heat.Add 0.5mL water come separated water layer and organic layer and extract ethyl acetate.Ethyl acetate extraction is repeated 2 times again, and extract is concentrated and dried overnight under 0.070mbar in a vacuum.End-product is the Mg salt of (+)-cis IsoHopCO2N. ((+)-VIII) and a small amount of salt impurity.Be further purified and can to remove all (-)-trans IsoHopCO2N., carry out by epimerization, crystallization and/or complexation.

Step 2B: from (+)-.alpha.-bitter acid ((+)-IV) asymmetric synthesis (-)-cis IsoHopCO2N. ((-)-VIII).

1431mg (+)-.alpha.-bitter acid ((+)-IV) is also under agitation warming up to 80 ° of C with the dilution of 1mL water in the 4mL bottle of adding a cover.The MgSO that adds 273mg (0.6eq) ₄, and continue to stir 5 minutes.38.25% (w/v) KOH solution of 430 μ L (1.0eq) is added to this solution, and makes reaction proceed 6 hours under 80 ° of C, now through HPLC judgement, reacted.The product of gained can be used to produce free acid or the Mg salt of (-)-cis IsoHopCO2N. ((-)-VIII).In this case, reactant mixture is remained in solution and by following steps 3B and be directly used in (-) KDT500.

By by the H of the water of the isopropyl alcohol of 2 volumes, 2 volumes and 1 equivalent ₂sO ₄be added to the free acid that forms (-)-cis IsoHopCO2N. ((-)-VIII) in reactant mixture.This mixture is mixed until complete and homogeneous is now carried out extractive reaction mixture (3x) with the dichloromethane of 2 volumes.Organic extract liquid is concentrated in a vacuum, then be dissolved in hexane, Na used ₂sO ₄dry, filter, and in a vacuum concentrated and under 0.070mbar dried overnight, the free acid of obtain (-)-cis IsoHopCO2N. ((-)-VIII).

By reactant mixture is removed and is under agitation added the ethyl acetate of 2 volumes until dissolve the Mg salt that forms (-)-cis IsoHopCO2N. ((-)-VIII) completely from heat.Add the water of 2 volumes come separated water layer and organic layer and extract ethyl acetate.The extraction of this ethyl acetate is repeated 2 times again, and by extract in a vacuum concentrated and under 0.070mbar dried overnight, the Mg salt of obtain (-)-cis IsoHopCO2N. ((-)-VIII) and a small amount of salt impurity.Be further purified and can pass through epimerization, crystallization, chromatograph, and/or complexation is carried out to remove all (+)-trans IsoHopCO2N..

Step 3A: by synthetic (+)-KDT500 (RTP:K008-50) of (+)-cis IsoHopCO2N. ((+)-VIII)

The dry Mg salt of 96.3mg (+)-cis IsoHopCO2N. ((+)-VIII) is dissolved in 1mL methanol.The MgO that adds 3.1mg (0.3eq), and stirred reaction mixture 5 minutes.The 10%Pd/C of 30mg is added in solution, and continues again to stir.By being advertised, hydrogen in solution, imports nitrogen atmosphere.After 45 minutes, through HPLC judgement, reacted, stop stirring, and remove nitrogen atmosphere by opening bottle.The sulphuric acid of 1 equivalent is added to reactant mixture (pH～1), and filters reaction by 0.2mm syringe filter.Reaction vessel and filter are one after the other washed 2 times by ethyl acetate.Filtrate is concentrated in a vacuum, then be dissolved in hexane, Na used ₂sO ₄dry, by Cotton Gossypii suction pipe, filter, and concentrate in a vacuum and dried overnight under 0.070mbar.Produce (+)-KDT-500 of 58.1mg.By chromatograph and crystallization, be further purified.

Step 3B: by synthetic (-)-KDT500 of (-)-cis IsoHopCO2N. ((-)-VIII).

The reactant mixture of step 2B ((-) of～1.4g-cis IsoHopCO2N .) is dissolved in 10mL methanol, adds the MgO of 256mg stirred reaction mixture 5 minutes.By this solution filter and be divided into 2 parts of isopyknic storing solutions.The MgO of 34mg is added to " reacting 1 ", then adds the 10%Pd/C of 200mg, then continue to stir.By being blasted, hydrogen in solution, imports nitrogen atmosphere.After 1 hour, by HPLC, judge and reacted, stop stirring, by opening bottle, discharge hydrogen.The sulphuric acid of 1 equivalent is added to reactant mixture (pH～1), and reaction is filtered by 0.2mm syringe type filter.Use continuously ethyl acetate washing reaction container and filter 2 times.Filtrate is concentrated in a vacuum, then be dissolved in hexane, Na used ₂sO ₄dry, by Cotton Gossypii suction pipe, filter, concentrated in a vacuum, and under 0.070mbar dried overnight.After merging two reactions, produce (-)-KDT-500 of 933.5mg.By chromatograph and crystallization, be further purified.

embodiment 13: from (-)-.alpha.-bitter acid and deoxidation .alpha.-bitter acid, prepare respectively (+)-.alpha.-bitter acid and (-)-.alpha.-bitter acid:

Diagram IV

Step 1: raceme (±)-.alpha.-bitter acid (III) synthetic.

Optional 1A: from synthetic raceme (±)-.alpha.-bitter acid (III) of (-)-.alpha.-bitter acid ((-)-IV).

Pure (-)-.alpha.-bitter acid is dissolved in to firpene or other applicable solvents (inertia high boiling alkane, for example, limonene, trimethyl cyclohexane) and forms 3-10% solution.This solution is placed in to manometer tube, with argon or nitrogen purging to remove all oxygen, sealing, and at 125 to 145 ° of C heating 1-20 hour.Then make reaction be cooled to room temperature, in a vacuum except desolventizing, if desired, and according to stating in other part places, purification raceme (±)-.alpha.-bitter acid (III).

Optional 1B: from synthetic raceme (±)-.alpha.-bitter acid (III) of deoxidation .alpha.-bitter acid (I).

Deoxidation .alpha.-bitter acid (I) is dissolved in methanol completely, and the lead acetate of 1eq is added to this solution, then add (0.1eq) 10%Pd/C of catalytic amount.Solution stirring is also used to air bubbling.After approximately 4 hours, by isolated by filtration lead salt, and extra lead acetate is added in filtrate to guarantee reclaiming raceme (±)-.alpha.-bitter acid (III) completely with the form of lead salt.All lead salts (foresythia) are collected and are suspended in methanol and then sulphuric acid is added in solution.Lead sulfate forms insoluble precipitation, by centrifugal, is removed.By the extraction between hexane and water (each 2 volumes roughly) of the methanol solution of homogeneous.Raceme (±)-.alpha.-bitter acid (III) is extracted in hexane and concentrates in a vacuum the free acid form that obtains raceme (±)-.alpha.-bitter acid (III).

Step 2A: from raceme (±)-.alpha.-bitter acid (III) purification (-)-.alpha.-bitter acid ((-)-IV).

Prepare respectively the solution of raceme (±)-.alpha.-bitter acid (III) in methanol (10%, w/v) and 1R, 2R-4-cyclohexene-1, the solution of 2-diamidogen in methanol (10%, w/v).These two kinds of solution are mixed with equimolar amounts (about 3:1 by volume), evaporation methanol, and residue is dissolved in again for example, in the 2-propanol (can use other solvents, t-butyl methyl ether, acetonitrile, ethyl acetate or benzene) of minimum flow.Form crystallization, by the amine salt of (-)-.alpha.-bitter acid (IV), formed, and (+)-.alpha.-bitter acid ((+)-IV) salt is retained in solution.Crystallization is filtered, be dried, then mix to discharge the free acid of (-)-.alpha.-bitter acid ((-)-IV) with hexane and aq. sulphuric acid.Hexane layer is separated, use salt water washing, by dried over sodium sulfate, and evaporation obtains (-)-.alpha.-bitter acid ((-)-IV) as shown in chirality HPLC.

Step 2B: from raceme (±)-.alpha.-bitter acid (III) purification (+)-.alpha.-bitter acid ((+)-IV)

Prepare respectively the solution of raceme (±)-.alpha.-bitter acid (III) in methanol (10%, w/v) and 1S, 2S-4-cyclohexene-1, the solution of 2-diamidogen in methanol (10%, w/v).These two kinds of solution are mixed with equimolar amounts (about 3:1 by volume), evaporation methanol, and residue is dissolved in again for example, in the 2-propanol (can use other solvents, t-butyl methyl ether, acetonitrile, ethyl acetate or benzene) of minimum flow.Can form crystallization, by the amine salt of (+)-.alpha.-bitter acid ((+)-IV), formed, and (-)-.alpha.-bitter acid ((-)-IV) salt be retained in solution.Crystallization is filtered, be dried, then mix to discharge the free acid of (+)-.alpha.-bitter acid ((+)-IV) with hexane and aq. sulphuric acid.Hexane layer is separated, use salt water washing, by dried over sodium sulfate, and evaporation obtains (+)-.alpha.-bitter acid ((+)-IV) as shown in chirality HPLC.

embodiment 14: from tetrahydrochysene deoxidation .alpha.-bitter acid preparation (+)-tetrahydrochysene .alpha.-bitter acid and (-)-tetrahydrochysene .alpha.-bitter acid:

Diagram V

Step 1: from synthetic raceme (±)-tetrahydrochysene .alpha.-bitter acid (IX) of tetrahydrochysene deoxidation .alpha.-bitter acid (VI).

Preparation tetrahydrochysene deoxidation .alpha.-bitter acid (VI) 10% (w/v) solution in acetic acid, and the concentrated sulphuric acid of catalytic amount is added in solution.Agitating solution, and drip 30% (w/w) hydrogenperoxide steam generator of 1eq.After 2 hours, with HPLC judgement, reacted, then add water and dichloromethane.Raceme (±)-tetrahydrochysene .alpha.-bitter acid (IX) is extracted in dichloromethane and concentrates in a vacuum the free acid form that obtains raceme (±)-tetrahydrochysene .alpha.-bitter acid (IX).

Step 2A: from raceme (±)-tetrahydrochysene .alpha.-bitter acid (IX) purification (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI).

Prepare respectively the solution of raceme (±)-tetrahydrochysene .alpha.-bitter acid (IX) in methanol (10%, w/v) and 1R, 2R-4-cyclohexene-1, the solution of 2-diamidogen in methanol (10%, w/v).These two kinds of solution are mixed with equimolar amounts (about 3:1 by volume), evaporation methanol, and residue is dissolved in again for example, in the 2-propanol (can use other solvents, t-butyl methyl ether, acetonitrile, ethyl acetate or benzene) of minimum flow.Can form crystallization, by the amine salt of (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI), formed, and (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI) salt be retained in solution.Crystallization is filtered, be dried, then mix to discharge the free acid of (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI) with hexane and aq. sulphuric acid.Hexane layer is separated, use salt water washing, by dried over sodium sulfate, and be evaporated to (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI) as shown in chirality HPLC.

Step 2B: from raceme (±)-tetrahydrochysene .alpha.-bitter acid purification (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI)

Prepare respectively the solution of raceme (±)-tetrahydrochysene .alpha.-bitter acid (IX) in methanol (10%, w/v) and 1S, 2S-4-cyclohexene-1, the solution of 2-diamidogen in methanol (10%, w/v).These two kinds of solution are mixed with equimolar amounts (about 3:1 by volume), evaporate methanol, residue is dissolved in to the 2-propanol (can use other solvents, for example methyl tertiary butyl ether(MTBE), acetonitrile, ethyl acetate or benzene) of minimum flow again.Can form crystallization, by the amine salt of (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI), formed, and (-)-tetrahydrochysene .alpha.-bitter acid ((-)-XI) salt can be retained in solution.Crystallization is filtered, be dried, then mix to discharge the free acid of (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI) with hexane and aq. sulphuric acid.Hexane layer is separated, use salt water washing, by dried over sodium sulfate, and be evaporated to (+)-tetrahydrochysene .alpha.-bitter acid ((+)-XI) as shown in HPLC.

embodiment 15: prepare other KDT500 salt:

Prepare calcium (II) salt (" KDT505 "):

The potassium salt (88.2mg) of water (1000 μ L) and KDT500 is added in 4mL bottle, then adds 26.6mg calcium chloride (1.10eq) and 120 μ L water.Mixture is stirred 1 hour and by sedimentation and filtration, with 1mL water washing 4x, then under fine vacuum, be dried and obtain 6.9mg product.This product has the fusing point of 125.1 ° of C and the water content of 1.28KF.

Prepare magnesium (II) salt (" KDT506 "):

The potassium salt (50.1mg) of water (1200 μ L) and KDT500 is added to 4mL bottle, then 7.8mg magnesium sulfate (0.53eq) and 200 μ L water.This mixture is stirred 1 hour and by sedimentation and filtration, with 1mL water washing 2x, and under fine vacuum, be dried acquisition 47mg product.This product has the fusing point of 130.0 ° of C and the water content of 1.27KF.

Prepare zinc (II) salt (" KDT507 "):

The potassium salt (84.7mg) of water (1300 μ L) and KDT500 is added in 4mL bottle, then adds 31.6mg Zinc vitriol (0.53eq) and 300 μ L water.This mixture is stirred 1 hour and by sedimentation and filtration, with 1mL water washing 2x, and under fine vacuum, be dried acquisition 71.6mg product.This product has the fusing point of 134.7 ° of C and the water content of 2.42KF.

Prepare ferrum (III) salt (" KDT508 "):

The potassium salt (61.9mg) of water (1000 μ L) and KDT500 is added in 4mL bottle, then adds 43.4mg ferric chloride hexahydrate (III) (0.53eq) He 300 μ L water.Mixture is stirred 1 hour and by sedimentation and filtration, with 1mL water washing 2x, and under fine vacuum, be dried acquisition 71.6mg product.This product has the fusing point of 72.9 ° of C and the water content of 1.07KF.

Prepare sodium (I) salt (" KDT509 "):

Water (1000 μ L) and KDT500 free salt (109.5mg) are added in 4mL bottle, then add 1M ag.NaOH solution (300 μ L, 1.00eq).By this mixture evaporation dry under fine vacuum.This product has the fusing point of 113.9 ° of C and the water content of 1.15KF.

Prepare copper (II) salt (" KDT510 "):

Water (1000 μ L) and KDT500 free salt (109.5mg) are added in 4mL bottle, then add 1M ag.NaOH solution (300 μ L, 1.00eq).By this mixture evaporation dry under fine vacuum.This product has the fusing point of 104.9 ° of C and the water content of 1.18KF.

Prepare guanidinesalt (" KDT511 "):

Water (1000 μ L) and KDT500 free salt (109.5mg) are added in 4mL bottle, then add 1M ag.NaOH solution (300 μ L, 1.00eq).By this mixture evaporation dry under fine vacuum.This product has the fusing point of 140.8 ° of C and the water content of 0.94KF.

As mentioned above, be only intended to illustrate various embodiment of the present invention above.Above-described concrete change should not be construed as limitation of the scope of the invention.To those skilled in the art, obviously can carry out without departing from the present invention various equivalents, variation and modification, and should understand so equivalent embodiment and should be encompassed in herein.All documents of quoting are herein incorporated to by quoting as described in detail completely in this article.

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Table 1: the crystallographic data of the structure providing

Table 2: the atomic coordinates (* 10 of crystallization (+)-KDT501 ⁴) and effective homogeneity displacement parameter

(U (eq) is defined as orthogonalized U ^ijthe track of tensor 1/3)

Table 3: the bond distance of crystallization (+)-KDT501 [

] and angle [] (being used for producing the symmetry transformation of equivalent atom: #1-x+3/2, y+1/2 ,-z+2#2-x+3/2, y-1/2 ,-z+2)

Table 4: the anisotropy displacement parameter of crystallization (+)-KDT501

Claims

1. (+)-(4S, the 5R)-3 with following structure, 4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone, or its salt or crystallization:

2. (+) as claimed in claim 1-(4S; 5R)-3; 4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles the salt of penta-2-alkene-1-ketone, the group that wherein said salt selects free potassium salt, aluminum salt, calcium salt, mantoquita, guanidinesalt, iron salt, lithium salts, magnesium salt, sodium salt, zinc salt, cinchonidine salt, cinchonine salt and diethanolamine salt to form.

3. the salt of claim 2, wherein said salt is the potassium salt with following structure:

4. the crystallization of the potassium salt of claim 3, wherein said crystallization has monoclinic system space group P21212, and unit cell dimension

α=90 °,

β=90 °,

and γ=90 °.

5. the crystallization of claim 4, wherein said crystallization has the three-dimensional atomic coordinates shown in table 2.

6. (+)-(4S as described in claim 1; 5R)-3,4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles the compositions of the enantiomer-pure substantially of penta-2-alkene-1-ketone or its salt or crystallization.

7. (+)-(4S as described in claim 1; 5R)-3,4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles the pharmaceutical compositions of the enantiomer-pure substantially of penta-2-alkene-1-ketone or its salt or crystallization and pharmaceutically acceptable carrier.

8. the pharmaceutical compositions of claim 7, the crystallization that it comprises claim 4.

9. treatment has a method for the disease that modulation responds to PPAR γ in this object needing, and it comprises to the pharmaceutical compositions of the claim 7 of described object drug treatment effective dose.

10. the method for claim 9, the group that wherein said disease selects free diabetes and obesity to form.

11. 1 kinds of treatments have the method for the disease that modulation responds to GPR120 in this object needing, and it comprises to the pharmaceutical compositions of the claim 7 of described object drug treatment effective dose.

12. 1 kinds of treatments have the method for the dysbolismus in this object needing, and it comprises to the pharmaceutical compositions of the claim 7 of described object drug treatment effective dose.

The method of 13. claim 12, wherein said dysbolismus is diabetes.

The method of 14. claim 12, the administration of wherein said pharmaceutical compositions causes glucose and/or lipid level to decline.

15. 1 kinds of inhibition have the method for the inflammation in this object needing, and it comprises to the pharmaceutical compositions of the claim 7 of described object drug treatment effective dose.

16. 1 kinds of treatments have the method for the disease relevant to inflammation in this object needing, and it comprises to the pharmaceutical compositions of the claim 7 of described object drug treatment effective dose.

17. crystallization (+)-(4S, 5R)-3,4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone potassium salt (1:1).

18. crystallization (-)-(4R, 5S)-3,4-dihydroxy-2-(3-methylbutyryl base)-5-(3-methyl butyl)-4-(4-methylpent acyl group) encircles penta-2-alkene-1-ketone potassium salt (1:1).